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Sample records for human hcn4 channels

  1. Effects of Shensong Yangxin capsule on pacemaker channels encoded by human HCN4 gene

    Institute of Scientific and Technical Information of China (English)

    SUN Li-ping; LI Ning; WU Yi-ling; PU Jie-lin

    2010-01-01

    @@ Shensong Yangxin (SSYX) is one of the compound recipes of Chinese materia medica including 12ingredients such as Panax ginseng, dwarf lilyturf tuber,nardostachys root, etc. Small-scale randomized multi-centre clinical trials suggested that SSYX reduced the number of ventricular extrasystoles in patients with or without structural heart disease.1 Besides excellent antiarrhythmic efficacy,2 SSYX also improved bradycardia in some patients, which was evidenced by animal studies3 as well. However, the antiarrhythmic mechanisms of SSYX have not been fully understood.Our previous studies have explored effect of SSYX on many channels except hyperpolarization-activated cation channel encoded by human hHCN4 gene.4

  2. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart

    Science.gov (United States)

    Stieber, Juliane; Herrmann, Stefan; Feil, Susanne; Löster, Jana; Feil, Robert; Biel, Martin; Hofmann, Franz; Ludwig, Andreas

    2003-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with “primitive” pacemaker action potentials could be found. However, cardiac cells with “mature” pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node. PMID:14657344

  3. Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function.

    Science.gov (United States)

    Biel, Stephanie; Aquila, Marco; Hertel, Brigitte; Berthold, Anne; Neumann, Thomas; DiFrancesco, Dario; Moroni, Anna; Thiel, Gerhard; Kauferstein, Silke

    2016-10-01

    Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality.

  4. Identification of the molecular site of ivabradine binding to HCN4 channels.

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    Annalisa Bucchi

    Full Text Available Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the "open channel block", the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.

  5. Pacemaker Activity of the Human Sinoatrial Node: An Update on the Effects of Mutations in HCN4 on the Hyperpolarization-Activated Current

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    Arie O. Verkerk

    2015-01-01

    Full Text Available Since 2003, several loss-of-function mutations in the HCN4 gene, which encodes the HCN4 protein, have been associated with sinus node dysfunction. In human sinoatrial node (SAN, HCN4 is the most abundant of the four isoforms of the HCN family. Tetramers of HCN subunits constitute the ion channels that conduct the hyperpolarization-activated “funny” current (If, which plays an important modulating role in SAN pacemaker activity. Voltage-clamp experiments on HCN4 channels expressed in COS-7, CHO and HEK-293 cells, as well as in Xenopus oocytes have revealed changes in the expression and kinetics of mutant channels, but the extent to which especially the kinetic changes would affect If flowing during a human SAN action potential often remains unresolved. In our contribution to the Topical Collection on Human Single Nucleotide Polymorphisms and Disease Diagnostics, we provide an updated review of the mutation-induced changes in the expression and kinetics of HCN4 channels and provide an overview of their effects on If during the time course of a human SAN action potential, as assessed in simulated action potential clamp experiments. Future research may solve apparent inconsistencies between data from clinical studies and data from in vitro and in silico experiments.

  6. The HCN4 channel mutation D553N associated with bradycardia has a C-linker mediated gating defect.

    Science.gov (United States)

    Netter, Michael F; Zuzarte, Marylou; Schlichthörl, Günter; Klöcker, Nikolaj; Decher, Niels

    2012-01-01

    The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N. Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N. Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant. The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel. Copyright © 2012 S. Karger AG, Basel.

  7. A novel mutation in the HCN4 gene causes symptomatic sinus bradycardia in Moroccan Jews.

    Science.gov (United States)

    Laish-Farkash, Avishag; Glikson, Michael; Brass, Dovrat; Marek-Yagel, Dina; Pras, Elon; Dascal, Nathan; Antzelevitch, Charles; Nof, Eyal; Reznik, Haya; Eldar, Michael; Luria, David

    2010-12-01

    to conduct a clinical, genetic, and functional analysis of 3 unrelated families with familial sinus bradycardia (FSB). mutations in the hyperpolarization-activated nucleotide-gated channel (HCN4) are known to be associated with FSB. three males of Moroccan Jewish descent were hospitalized: 1 survived an out-of-hospital cardiac arrest and 2 presented with weakness and presyncopal events. All 3 had significant sinus bradycardia, also found in other first-degree relatives, with a segregation suggesting autosomal-dominant inheritance. All had normal response to exercise and normal heart structure. Sequencing of the HCN4 gene in all patients revealed a C to T transition at nucleotide position 1,454, which resulted in an alanine to valine change (A485V) in the ion channel pore found in most of their bradycardiac relatives, but not in 150 controls. Functional expression of the mutated ion channel in Xenopus oocytes and in human embryonic kidney 293 cells revealed profoundly reduced function and synthesis of the mutant channel compared to wild-type. we describe a new mutation in the HCN4 gene causing symptomatic FSB in 3 unrelated individuals of similar ethnic background that may indicate unexplained FSB in this ethnic group. This profound functional defect is consistent with the symptomatic phenotype.

  8. Research Progress on Pace-making Gene HCN4 of Sinoatrial Node Cells%窦房结细胞起搏基因HCN4的研究进展

    Institute of Scientific and Technical Information of China (English)

    王妮娜

    2011-01-01

    HCN即超级化激活的环核苷酸门控阳离子通道,其激活后产生的If/Ih离子流是窦房结起搏细胞动作电位正常形成的分子基础.随着对窦房结细胞起搏机制和HCN基因家族研究的不断深入,人们对HCN亚型HCN4的结构、分布、特性已有了较深入的了解.近年来有较多研究表明,人窦房结起搏基因HCN4突变与病态窦房结综合征密切相关.现就窦房结细胞起搏基因HCN4的特性及其与窦房结功能之间的关系作进一步研究和探讨.%Hyperpolarization-activated cyclic nucleotide-gated channel, also called HCN , is activated to release If/Ih currents that underlie the molecular mechanisms of action potential in sinoatrial node ( SAN)pace-making cells. The further investigations into the pace-making of SAN and HCN gene family allows the understanding of the structure, distribution, and property of HCN subtype 4 ( HCN4 ). Recent studies show that human SAN pace-making gene HCN4 mutations are closely associated with sick sinus syndrome. This article mainly reviews the features of SAN pace-making gene HCN4 in relation to SAN function.

  9. Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.

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    Yukihiro Saito

    Full Text Available Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN channels and attenuation of the inward rectifier K+ current (IK1 flowing through inward rectifier potassium (Kir channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs function as a biological pacemaker in vitro.The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded

  10. Effects of Yiqi Tongyang on HCN4 Protein Phosphorylation in Damaged Rabbit Sinoatrial Node Cells

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    Jinfeng Liu

    2016-01-01

    Full Text Available The hyperpolarization-activated cyclic nucleotide-gated cation channel (If is closely associated with sinoatrial node pacing function. The present study aimed to investigate the molecular mechanisms involved in pacing function improvements of damaged sinoatrial node cells and the consequent treatment effects on sick sinus syndrome (SSS after the use of Yiqi Tongyang. HCN4 channel protein expression and phosphorylation were measured by immunoblotting and fluorescent quantitation. After ischemia-reperfusion injury (model group, the HCN4 protein and the optical density (OD of the phosphorylated HCN4 protein as well as intracellular PKA activity in the sinoatrial node cells decreased significantly. However, the OD values and PKA activity increased to different degrees after treatment with serum containing different doses of Yiqi Tongyang; in contrast, no significant improvement was seen in the control group compared to the model group. These findings demonstrated that the use of the traditional Chinese medicine Yiqi Tongyang could increase HCN4 protein expression and phosphorylation as well as PKA activity within sinoatrial node cells damaged by ischemia-reperfusion. The HCN4 protein is involved in the If-related ion channel. Here, we speculated that these effects could be associated with upregulation of HCN4 protein phosphorylation, which consequently improved cell automaticity, increased heart rate, and had treatment effects on SSS.

  11. Effects of Yiqi Tongyang on HCN4 Protein Phosphorylation in Damaged Rabbit Sinoatrial Node Cells.

    Science.gov (United States)

    Liu, Jinfeng; Liu, Ruxiu; Peng, Jie; Wang, Yanli

    2016-01-01

    The hyperpolarization-activated cyclic nucleotide-gated cation channel (I f ) is closely associated with sinoatrial node pacing function. The present study aimed to investigate the molecular mechanisms involved in pacing function improvements of damaged sinoatrial node cells and the consequent treatment effects on sick sinus syndrome (SSS) after the use of Yiqi Tongyang. HCN4 channel protein expression and phosphorylation were measured by immunoblotting and fluorescent quantitation. After ischemia-reperfusion injury (model group), the HCN4 protein and the optical density (OD) of the phosphorylated HCN4 protein as well as intracellular PKA activity in the sinoatrial node cells decreased significantly. However, the OD values and PKA activity increased to different degrees after treatment with serum containing different doses of Yiqi Tongyang; in contrast, no significant improvement was seen in the control group compared to the model group. These findings demonstrated that the use of the traditional Chinese medicine Yiqi Tongyang could increase HCN4 protein expression and phosphorylation as well as PKA activity within sinoatrial node cells damaged by ischemia-reperfusion. The HCN4 protein is involved in the I f -related ion channel. Here, we speculated that these effects could be associated with upregulation of HCN4 protein phosphorylation, which consequently improved cell automaticity, increased heart rate, and had treatment effects on SSS.

  12. Novel pharmacological activity of loperamide and CP-339,818 on human HCN channels characterized with an automated electrophysiology assay.

    Science.gov (United States)

    Lee, Yan T; Vasilyev, Dmitry V; Shan, Qin J; Dunlop, John; Mayer, Scott; Bowlby, Mark R

    2008-02-26

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons (I(h)) and cardiac (I(f)) cells. As such, the identification and characterization of novel blockers of HCN channels is important to enable the dissection of their function in vivo. Using a new IonWorks HT electrophysiology assay with human HCN1 and HCN4 expressed stably in cell lines, four HCN channel blockers are characterized. Two blockers known for their activity at opioid/Ca(2+) channels and K(+) channels, loperamide and CP-339,818 (respectively), are described to block HCN1 more potently than HCN4. The known HCN blocker ZD7288 was also found to be more selective for HCN1 over HCN4, while the HCN blocker DK-AH269 was equipotent on HCN4 and HCN1. Partial replacement of the intracellular Cl(-) with gluconate reduced the potency on both channels, but to varying degrees. For both HCN1 and HCN4, ZD7288 was most sensitive in lower Cl(-) solutions, while the potency of loperamide was not affected by the differing solutions. The block of HCN1 for all compounds was voltage-dependent, being relieved at more negative potentials. The voltage-dependent, Cl(-) dependent, HCN1 preferring compounds described here elaborate on the current known pharmacology of HCN channels and may help provide novel tools and chemical starting points for the investigation of HCN channel function in natively expressing systems.

  13. HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus

    Science.gov (United States)

    Hughes, D.I.; Boyle, K.A.; Kinnon, C.M.; Bilsland, C.; Quayle, J.A.; Callister, R.J.; Graham, B.A.

    2013-01-01

    Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1–4), which confer distinct biophysical properties on the channel. Despite understanding the structure–function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several

  14. HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus.

    Science.gov (United States)

    Hughes, D I; Boyle, K A; Kinnon, C M; Bilsland, C; Quayle, J A; Callister, R J; Graham, B A

    2013-05-01

    Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1-4), which confer distinct biophysical properties on the channel. Despite understanding the structure-function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several

  15. Pacemaker current inhibition in experimental human cardiac sympathetic activation: a double-blind, randomized, crossover study

    NARCIS (Netherlands)

    Schroeder, C.; Heusser, K.; Zoerner, A.A.; Grosshennig, A.; Wenzel, D.; May, M.; Sweep, F.C.; Mehling, H.; Luft, F.C.; Tank, J.; Jordan, J.

    2014-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated 4 (HCN4) channels comprise the final pathway for autonomic heart rate (HR) regulation. We hypothesized that HCN4 inhibition could reverse autonomic imbalance in a human model of cardiac sympathetic activation. Nineteen healthy men ingested oral me

  16. Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells.

    Science.gov (United States)

    Feng, Yuanyuan; Luo, Shouming; Yang, Pan; Song, Zhiyuan

    2016-04-01

    The 'funny' current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was -101.2±4.6 mV and the time constant of activation was 324±41 msec under -160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to -92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels may have a

  17. 兔特发性室性心动过速心室流出道HCN4蛋白表达的实验观察%Expression of HCN4 protein in ventricular outflow tract of rabbit with idiopathic ventricular tachycardia

    Institute of Scientific and Technical Information of China (English)

    吴晓羽; 姜晓慧; 李怀娜; 田平; 周中华; 时珊珊; 于静; 丁玲玲

    2015-01-01

    目的 探讨超极化激活的环核苷酸门控离子通道亚型4 (HCN4)蛋白在室性心动过速(室速)中的作用.方法 数字随机选择新西兰大白兔10只,应用HE染色及免疫组织化学检测中间神经纤维丝(NF-M),观察心室流出道组织是否存在浦肯野纤维.另选择新西兰大白兔40只平均分4组即对照组(SO)、室速组(ⅤT)、室速+艾司洛尔干预组(ⅤT+ ESM)、室速+伊伐布雷定干预组(ⅤT+ⅣA).采用免疫组化方法检测心室流出道HCN4蛋白的表达,观察并记录各组诱发室速时所需电压的输出幅值、停止刺激后自发性室速发生的次数及持续时间.结果 (1)兔心室流出道存在浦肯野纤维.(2)左、右心室流出道HCN4蛋白的表达ⅤT组与SO组比较明显增多(左室:97.6±16.7与29.0±8.0,P<0.01;右室:92.7±12.3与26.0±10.8,P<0.01);ⅤT+ ⅣA组与ⅤT组比较明显减少(左室:32.0±9.4与97.6±16.7,P<0.01;右室:30.8±12.4与92.7±12.3,P<0.01).(3)ⅤT +ESM组和ⅤT+ ⅣA组与YT组相比,在同等高频刺激的条件下诱发流出道室速所需的输出电压幅值增加(P<0.01),停止刺激后自发性室速的发生次数减少(P<0.01),自发性室速持续时间缩短(P<0.01).结论 (1)心室流出道存在浦肯野纤维,且室速发生时HCN4蛋白表达上调;(2)艾司洛尔及伊伐布雷定可以预防及减少室速的发生,伊伐布雷定作为HCN通道的特异性通道抑制剂效果更强.%Objective To confirm the existence of purkinje fibers in rabbit outflow tract tissue and explore the role of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 4 (HCN4) protein in idiopathic ventricular tachycardia.Methods A total of ten New Zealand white rabbits were randomly selected to observe whether there were pukinje fibers in outflow tract by the methods of HE staining and immunohistochemical detection of midsize neurofilament (NF-M).Forty rabbits were randomly divided into four groups : normal control group (SO

  18. A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

    Science.gov (United States)

    Hategan, Lidia; Csányi, Beáta; Ördög, Balázs; Kákonyi, Kornél; Tringer, Annamária; Kiss, Orsolya; Orosz, Andrea; Sághy, László; Nagy, István; Hegedűs, Zoltán; Rudas, László; Széll, Márta; Varró, András; Forster, Tamás; Sepp, Róbert

    2017-08-15

    The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rateheart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. TRP Channels in Human Prostate

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    Carl Van Haute

    2010-01-01

    Full Text Available This review gives an overview of morphological and functional characteristics in the human prostate. It will focus on the current knowledge about transient receptor potential (TRP channels expressed in the human prostate, and their putative role in normal physiology and prostate carcinogenesis. Controversial data regarding the expression pattern and the potential impact of TRP channels in prostate function, and their involvement in prostate cancer and other prostate diseases, will be discussed.

  20. HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus

    OpenAIRE

    Hughes, D.I.; Boyle, K.A.; Kinnon, C.M.; Bilsland, C.; Quayle, J A; Callister, R. J.; Graham, B.A.

    2013-01-01

    Hyperpolarisation-activated (Ih ) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1–4), which confer distinct biophysical properties on the channel. Despite understanding the struct...

  1. Rescue of a trafficking defective human pacemaker channel via a novel mechanism: roles of Src, Fyn, and Yes tyrosine kinases.

    Science.gov (United States)

    Lin, Yen-Chang; Huang, Jianying; Kan, Hong; Frisbee, Jefferson C; Yu, Han-Gang

    2009-10-30

    Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.

  2. ASTE Simultaneous HCN(4-3) and HCO+(4-3) Observations of the Two Luminous Infrared Galaxies NGC 4418 and Arp 220

    CERN Document Server

    Imanishi, Masatoshi; Yamada, Masako; Tamura, Yoichi; Kohno, Kotaro

    2010-01-01

    We report the results of HCN(J=4-3) and HCO+(J=4-3) observations of two luminous infrared galaxies (LIRGs), NGC 4418 and Arp 220, made using the Atacama Submillimeter Telescope Experiment (ASTE). The ASTE wide-band correlator provided simultaneous observations of HCN(4-3) and HCO+(4-3) lines, and a precise determination of their flux ratios. Both galaxies showed high HCN(4-3) to HCO+(4-3) flux ratios of >2, possibly due to AGN-related phenomena. The J = 4-3 to J = 1-0 transition flux ratios for HCN (HCO+) are similar to those expected for fully thermalized (sub-thermally excited) gas in both sources, in spite of HCN's higher critical density. If we assume collisional excitation and neglect an infrared radiative pumping process, our non-LTE analysis suggests that HCN traces gas with significantly higher density than HCO+. In Arp 220, we separated the double-peaked HCN(4-3) emission into the eastern and western nuclei, based on velocity information. We confirmed that the eastern nucleus showed a higher HCN(4-3)...

  3. Ivabradine prolongs phase 3 of cardiac repolarization and blocks the hERG1 (KCNH2) current over a concentration-range overlapping with that required to block HCN4.

    Science.gov (United States)

    Lees-Miller, James P; Guo, Jiqing; Wang, Yibo; Perissinotti, Laura L; Noskov, Sergei Y; Duff, Henry J

    2015-08-01

    In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the If current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance.

  4. Star Formation Laws in Both Galactic Massive Clumps and External Galaxies: Extensive Study with Dust Coninuum, HCN (4-3), and CS (7-6)

    Science.gov (United States)

    Liu, Tie; Kim, Kee-Tae; Yoo, Hyunju; Liu, Sheng-yuan; Tatematsu, Ken'ichi; Qin, Sheng-Li; Zhang, Qizhou; Wu, Yuefang; Wang, Ke; Goldsmith, Paul F.; Juvela, Mika; Lee, Jeong-Eun; Tóth, L. Viktor; Mardones, Diego; Garay, Guido; Bronfman, Leonardo; Cunningham, Maria R.; Li, Di; Lo, Nadia; Ristorcelli, Isabelle; Schnee, Scott

    2016-10-01

    We observed 146 Galactic clumps in HCN (4-3) and CS (7-6) with the Atacama Submillimeter Telescope Experiment 10 m telescope. A tight linear relationship between star formation rate and gas mass traced by dust continuum emission was found for both Galactic clumps and the high redshift (z > 1) star forming galaxies (SFGs), indicating a constant gas depletion time of ˜100 Myr for molecular gas in both Galactic clumps and high z SFGs. However, low z galaxies do not follow this relation and seem to have a longer global gas depletion time. The correlations between total infrared luminosities (L TIR) and molecular line luminosities ({L}{mol}\\prime ) of HCN (4-3) and CS (7-6) are tight and sublinear extending down to clumps with L TIR ˜ 103 L ⊙. These correlations become linear when extended to external galaxies. A bimodal behavior in the L TIR-{L}{mol}\\prime correlations was found for clumps with different dust temperature, luminosity-to-mass ratio, and σ line/σ vir. Such bimodal behavior may be due to evolutionary effects. The slopes of L TIR-L‧mol correlations become more shallow as clumps evolve. We compared our results with lower J transition lines in Wu et al. (2010). The correlations between clump masses and line luminosities are close to linear for low effective excitation density tracers but become sublinear for high effective excitation density tracers for clumps with L TIR larger than L TIR ˜ 104.5 L ⊙. High effective excitation density tracers cannot linearly trace the total clump masses, leading to a sublinear correlations for both M clump-L‧mol and L TIR-L‧mol relations.

  5. Pharmacology of the human red cell voltage-dependent cation channel Part I. Activation by clotrimazole and analogues

    DEFF Research Database (Denmark)

    Barksmann, Trine Lyberth; Kristensen, Berit I.; Christophersen, Palle.

    2004-01-01

    Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators......Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators...

  6. Potassium channels and human epileptic phenotypes: an updated overview

    Directory of Open Access Journals (Sweden)

    Chiara eVilla

    2016-03-01

    Full Text Available Potassium (K+ channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K+ channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K+ channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K+ channels dysfunctions linked to inherited epilepsy in humans and non-human model animals.This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K+ channels in monogenic forms.

  7. Box model for channels of human migration

    CERN Document Server

    Vitanov, Nikolay K

    2016-01-01

    We discuss a mathematical model of migration channel based on the truncated Waring distribution. The truncated Waring distribution is obtained for a more general model of motion of substance through a channel containing finite number of boxes. The model is applied then for case of migrants moving through a channel consisting of finite number of countries or cities. The number of migrants in the channel strongly depends on the number of migrants that enter the channel through the country of entrance. It is shown that if the final destination country is very popular then large percentage of migrants may concentrate there.

  8. DDESC: Dragon database for exploration of sodium channels in human

    Directory of Open Access Journals (Sweden)

    Radovanovic Aleksandar

    2008-12-01

    Full Text Available Abstract Background Sodium channels are heteromultimeric, integral membrane proteins that belong to a superfamily of ion channels. The mutations in genes encoding for sodium channel proteins have been linked with several inherited genetic disorders such as febrile epilepsy, Brugada syndrome, ventricular fibrillation, long QT syndrome, or channelopathy associated insensitivity to pain. In spite of these significant effects that sodium channel proteins/genes could have on human health, there is no publicly available resource focused on sodium channels that would support exploration of the sodium channel related information. Results We report here Dragon Database for Exploration of Sodium Channels in Human (DDESC, which provides comprehensive information related to sodium channels regarding different entities, such as "genes and proteins", "metabolites and enzymes", "toxins", "chemicals with pharmacological effects", "disease concepts", "human anatomy", "pathways and pathway reactions" and their potential links. DDESC is compiled based on text- and data-mining. It allows users to explore potential associations between different entities related to sodium channels in human, as well as to automatically generate novel hypotheses. Conclusion DDESC is first publicly available resource where the information related to sodium channels in human can be explored at different levels. This database is freely accessible for academic and non-profit users via the worldwide web http://apps.sanbi.ac.za/ddesc.

  9. Unsupervised Idealization of Ion Channel Recordings by Minimum Description Length: Application to Human PIEZO1-Channels

    DEFF Research Database (Denmark)

    Gnanasambandam, Radhakrishnan; Nielsen, Morten S; Nicolai, Christopher

    2017-01-01

    on inputs and supervision by the user, thus requiring some prior knowledge of underlying processes. Channels with unknown gating and/or functional sub-states and the presence in the recording of currents from uncorrelated background channels present substantial challenges to such analyses. Here we describe...... channel currents and their substates from recordings with multiple channels, even under conditions of high noise. We then tested the MDL algorithm on real experimental data from human PIEZO1 channels and found that our method revealed the presence of substates with alternate conductances....

  10. Human sperm cells swimming in micro-channels

    CERN Document Server

    Denissenko, Petr; Smith, David; Kirkman-Brown, Jackson

    2012-01-01

    The migratory abilities of motile human spermatozoa in vivo are essential for natural fertility, but it remains a mystery what properties distinguish the tens of cells which find an egg from the millions of cells ejaculated. To reach the site of fertilization, sperm must traverse narrow and convoluted channels, filled with viscous fluids. To elucidate individual and group behaviors that may occur in the complex three-dimensional female tract environment, we examine the behavior of migrating sperm in assorted micro-channel geometries. Cells rarely swim in the central part of the channel cross-section, instead traveling along the intersection of the channel walls (`channel corners'). When the channel turns sharply, cells leave the corner, continuing ahead until hitting the opposite wall of the channel, with a distribution of departure angles, the latter being modulated by fluid viscosity. If the channel bend is smooth, cells depart from the inner wall when the curvature radius is less than a threshold value clo...

  11. Pharmacology of the human cell voltage-dependent cation channel. Part II: inactivation and blocking

    DEFF Research Database (Denmark)

    Bennekou, Poul; Barksmann, Trine L.; Kristensen, Berit I.

    2004-01-01

    Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents......Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents...

  12. Recent genetic discoveries implicating ion channels in human cardiovascular diseases.

    Science.gov (United States)

    George, Alfred L

    2014-04-01

    The term 'channelopathy' refers to human genetic disorders caused by mutations in genes encoding ion channels or their interacting proteins. Recent advances in this field have been enabled by next-generation DNA sequencing strategies such as whole exome sequencing with several intriguing and unexpected discoveries. This review highlights important discoveries implicating ion channels or ion channel modulators in cardiovascular disorders including cardiac arrhythmia susceptibility, cardiac conduction phenotypes, pulmonary and systemic hypertension. These recent discoveries further emphasize the importance of ion channels in the pathophysiology of human disease and as important druggable targets.

  13. Human myoblast differentiation: Ca(2+) channels are activated by K(+) channels.

    Science.gov (United States)

    Bernheim, Laurent; Bader, Charles R

    2002-02-01

    In a paradigm of cellular differentiation, human myoblast fusion, we investigated how a Ca(2+) influx, indispensable for fusion, is triggered. We show how newly expressed Kir2.1 K(+) channels, via their hyperpolarizing effect on the membrane potential, generate a window Ca(2+) current (mediated by alpha 1H T-type Ca(2+) channels), which causes intracellular Ca(2+) to rise.

  14. Genetic variation in Hyperpolarization-activated cyclic nucleotide-gated (HCN channels and its relationship with neuroticism, cognition and risk of depression

    Directory of Open Access Journals (Sweden)

    Andrew Mark Mcintosh

    2012-07-01

    Full Text Available Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are encoded by four genes (HCN1-4 and, through activation by cyclic AMP (cAMP, represent a point of convergence for several psychosis risk genes. On the basis of positive preliminary data, we sought to test whether genetic variation in HCN1-4 conferred risk of depression or cognitive impairment in the Generation Scotland: Scottish Family Health Study. HCN1, HCN2, HCN3 and HCN4 were genotyped for 43 haplotype-tagging SNPs and tested for association with DSM-IV depression, neuroticism and a battery of cognitive tests assessing cognitive ability, memory, verbal fluency and psychomotor performance. No association was found between any HCN channel gene SNP and risk of depression, neuroticism or on any cognitive measure. The current study does not support a genetic role for HCN channels in conferring risk of depression or cognitive impairment in human subjects within the Scottish population.

  15. TRESK potassium channel in human T lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Miguel, Dénison Selene, E-mail: amurusk@hotmail.com [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico); García-Dolores, Fernando, E-mail: garciaddf@yahoo.com [Department of Pathology, Institute of Forensic Sciences, Av. Niños Héroes 130, Col. Doctores, C.P. 06720 Mexico, DF (Mexico); Rosa Flores-Márquez, María, E-mail: mariafo31@yahoo.com.mx [National Medical Center of Occident (CMNO) IMSS, Belisario Dominguez 735, Col. Independencia Oriente, C.P. 44340 Guadalajara, Jalisco (Mexico); Delgado-Enciso, Iván [University of Colima, School of Medicine, Av. Universidad 333, Col. Las Viboras, C.P. 28040 Colima (Mexico); Pottosin, Igor, E-mail: pottosin@ucol.mx [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico); Dobrovinskaya, Oxana, E-mail: oxana@ucol.mx [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico)

    2013-05-03

    Highlights: • TRESK (KCNK18) mRNA is present in different T lymphoblastic cell lines. • KCNK18 mRNA was not found in resting peripheral blood lymphocytes. • Clinical samples of T lymphoblastic leukemias and lymphomas were positive for TRESK. • TRESK in T lymphoblasts has dual localization, in plasma membrane and intracellular. -- Abstract: TRESK (TWIK-related spinal cord K{sup +}) channel, encoded by KCNK18 gene, belongs to the double-pore domain K{sup +} channel family and in normal conditions is expressed predominantly in the central nervous system. In our previous patch-clamp study on Jurkat T lymphoblasts we have characterized highly selective K{sup +} channel with pharmacological profile identical to TRESK. In the present work, the presence of KCNK18 mRNA was confirmed in T lymphoblastic cell lines (Jurkat, JCaM, H9) but not in resting peripheral blood lymphocytes of healthy donors. Positive immunostaining for TRESK was demonstrated in lymphoblastic cell lines, in germinal centers of non-tumoral lymph nodes, and in clinical samples of T acute lymphoblastic leukemias/lymphomas. Besides detection in the plasma membrane, intracellular TRESK localization was also revealed. Possible involvement of TRESK channel in lymphocyte proliferation and tumorigenesis is discussed.

  16. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte Bjørg

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  17. Molecular dynamics insights into human aquaporin 2 water channel.

    Science.gov (United States)

    Binesh, A R; Kamali, R

    2015-12-01

    In this study, the first molecular dynamics simulation of the human aquaporin 2 is performed and for a better understanding of the aquaporin 2 permeability performance, the characteristics of water transport in this protein channel and key biophysical parameters of AQP2 tetramer including osmotic and diffusive permeability constants and the pore radius are investigated. For this purpose, recently recovered high resolution X-ray crystal structure of` the human aquaporin 2 is used to perform twenty nanosecond molecular dynamics simulation of fully hydrated tetramer of this protein embedded in a lipid bilayer. The resulting water permeability characteristics of this protein channel showed that the water permeability of the human AQP2 is in a mean range in comparison with other human aquaporins family. Finally, the results reported in this research demonstrate that molecular dynamics simulation of human AQP2 provided useful insights into the mechanisms of water permeation and urine concentration in the human kidney.

  18. Molecular Structure of the Human CFTR Ion Channel.

    Science.gov (United States)

    Liu, Fangyu; Zhang, Zhe; Csanády, László; Gadsby, David C; Chen, Jue

    2017-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.

  19. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    Science.gov (United States)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  20. Changes in channel morphology over human time scales [Chapter 32

    Science.gov (United States)

    John M. Buffington

    2012-01-01

    Rivers are exposed to changing environmental conditions over multiple spatial and temporal scales, with the imposed environmental conditions and response potential of the river modulated to varying degrees by human activity and our exploitation of natural resources. Watershed features that control river morphology include topography (valley slope and channel...

  1. Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

    Science.gov (United States)

    Gnanasambandam, Radhakrishnan; Bae, Chilman; Gottlieb, Philip A; Sachs, Frederick

    2015-01-01

    Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

  2. Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Gnanasambandam

    Full Text Available Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2 form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1 for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

  3. Human and automatic speaker recognition over telecommunication channels

    CERN Document Server

    Fernández Gallardo, Laura

    2016-01-01

    This work addresses the evaluation of the human and the automatic speaker recognition performances under different channel distortions caused by bandwidth limitation, codecs, and electro-acoustic user interfaces, among other impairments. Its main contribution is the demonstration of the benefits of communication channels of extended bandwidth, together with an insight into how speaker-specific characteristics of speech are preserved through different transmissions. It provides sufficient motivation for considering speaker recognition as a criterion for the migration from narrowband to enhanced bandwidths, such as wideband and super-wideband.

  4. HCN Channels and Heart Rate

    Directory of Open Access Journals (Sweden)

    Ilaria Dentamaro

    2012-04-01

    Full Text Available Hyperpolarization and Cyclic Nucleotide (HCN -gated channels represent the molecular correlates of the “funny” pacemaker current (If, a current activated by hyperpolarization and considered able to influence the sinus node function in generating cardiac impulses. HCN channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. This channel family comprises four members: HCN1-4, but there is a general agreement to consider HCN4 as the main isoform able to control heart rate. This review aims to summarize advanced insights into the structure, function and cellular regulation of HCN channels in order to better understand the role of such channels in regulating heart rate and heart function in normal and pathological conditions. Therefore, we evaluated the possible therapeutic application of the selective HCN channels blockers in heart rate control.

  5. Noise analysis and single-channel observations of 4 pS chloride channels in human airway epithelia.

    OpenAIRE

    Duszyk, M; French, A S; Man, S F

    1992-01-01

    Apical membranes of human airway epithelial cells have significant chloride permeability, which is reduced in cystic fibrosis (CF), causing abnormal electrochemistry and impaired mucociliary clearance. At least four types of chloride channels have been identified in these cells, but their relative roles in total permeability and CF are unclear. Noise analysis was used to measure the conductance of chloride channels in human nasal epithelial cells. The data indicate that channels with a mean c...

  6. TRPM8, a versatile channel in human sperm.

    Directory of Open Access Journals (Sweden)

    Gerardo A De Blas

    Full Text Available BACKGROUND: The transient receptor potential channel (TRP family includes more than 30 proteins; they participate in various Ca(2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+ ([Ca(2+]i. Ca(2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature and antagonists (BCTC and capsazepine. Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM and 80% by BCTC (1.6 microM. Activation of TRPM8 either by temperature or menthol induced [Ca(2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM and BCTC (1.6 microM. However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8 in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

  7. TRPM8, a Versatile Channel in Human Sperm

    Science.gov (United States)

    Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

    2009-01-01

    Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

  8. Functional properties of human neuronal Kv11 channels

    DEFF Research Database (Denmark)

    Einarsen, Karoline; Calloe, Kirstine; Grunnet, Morten

    2009-01-01

    Kv11 potassium channels are important for regulation of the membrane potential. Kv11.2 and Kv11.3 are primarily found in the nervous system, where they most likely are involved in the regulation of neuronal excitability. Two isoforms of human Kv11.2 have been published so far. Here, we present...... a new splice variant that is present in human brain as demonstrated by reverse transcription PCR. Heterologous expression in Xenopus laevis oocytes revealed a 30-mV shift in the voltage dependence of activation to more depolarized potentials and slower activation together with faster deactivation...

  9. Dynamic Propagation Channel Characterization and Modeling for Human Body Communication

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2012-12-01

    Full Text Available This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC. In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000 were acquired from five volunteers. Various path gains caused by different locations and movements were quantified and the statistical distributions were estimated. In general, for a given reference threshold è = −10 dB, the maximum average level crossing rate of the HBC was approximately 1.99 Hz, the maximum average fade time was 59.4 ms, and the percentage of bad channel duration time was less than 4.16%. The HBC exhibited a fade depth of −4 dB at 90% complementary cumulative probability. The statistical parameters were observed to be centered for each propagation channel. Subsequently a Fritchman model was implemented to estimate the burst characteristics of the on-body fading. It was concluded that the HBC is motion-insensitive, which is sufficient for reliable communication link during motions, and therefore it has great potential for body sensor/area networks.

  10. Dynamic propagation channel characterization and modeling for human body communication.

    Science.gov (United States)

    Nie, Zedong; Ma, Jingjing; Li, Zhicheng; Chen, Hong; Wang, Lei

    2012-12-18

    This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC). In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000) were acquired from five volunteers. Various path gains caused by different locations and movements were quantified and the statistical distributions were estimated. In general, for a given reference threshold è = -10 dB, the maximum average level crossing rate of the HBC was approximately 1.99 Hz, the maximum average fade time was 59.4 ms, and the percentage of bad channel duration time was less than 4.16%. The HBC exhibited a fade depth of -4 dB at 90% complementary cumulative probability. The statistical parameters were observed to be centered for each propagation channel. Subsequently a Fritchman model was implemented to estimate the burst characteristics of the on-body fading. It was concluded that the HBC is motion-insensitive, which is sufficient for reliable communication link during motions, and therefore it has great potential for body sensor/area networks.

  11. Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

    Science.gov (United States)

    Yechikov, Sergey; Copaciu, Raul; Gluck, Jessica M; Deng, Wenbin; Chiamvimonvat, Nipavan; Chan, James W; Lieu, Deborah K

    2016-07-19

    Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016.

  12. Aquaporin water channels: molecular mechanisms for human diseases.

    Science.gov (United States)

    Agre, Peter; Kozono, David

    2003-11-27

    Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H3O+). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.

  13. Immunolocalization and expression of small-conductance calcium-activated potassium channels in human myometrium

    DEFF Research Database (Denmark)

    Rosenbaum, Sofia T; Svalø, Julie; Nielsen, Karsten;

    2012-01-01

    Small-conductance calcium-activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA...

  14. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  15. Localization and function of ATP-sensitive potassium channels in human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jens Jung; Kristensen, Michael; Hellsten, Ylva

    2003-01-01

    The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique...

  16. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  17. Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

    Institute of Scientific and Technical Information of China (English)

    Byung Joo Kim; Kyu Joo Park; Hyung Woo Kim; Seok Choi; Jae Yeoul Jun; In Youb Chang; Ju-Hong Jeon; Insuk So; Seon Jeong Kim

    2009-01-01

    AIM: To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 (TRPM7) in the human gastrointestinal (GI) tract. METHODS: Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajal (ICCs). RESULTS: The human GI tract generated slow electrical waves and had ICCs which functioned as pacemaker cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB (2-aminoethoxydiphenyl borate) and La3~+, TRPM7 channel blockers, inhibited the slow waves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION: These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the generation of the slow waves.

  18. Cloning and characterization of a human delayed rectifier potassium channel gene.

    Science.gov (United States)

    Albrecht, B; Lorra, C; Stocker, M; Pongs, O

    1993-01-01

    A human genomic DNA library was screened for sequences homologues to the rat delayed rectifier Kv 2.1 (DRK1) K+ channel cDNA. Three phages were isolated which hybridized to Kv 2.1 cDNA probes. Alignment of the human genomic DNA sequence with the rat cDNA sequence indicated that the open reading frame (ORF) is interrupted by a large intervening sequence, that separates exons encoding the membrane spanning core region of the K+ channel polypeptide. The Kv 2.1 gene occurs once in the human genome and has been mapped to chromosome 20. The human, mouse and rat Kv 2.1 proteins have been highly conserved, showing only a few substitutions outside of the membrane spanning domains in the amino- and carboxy-terminal cytoplasmic domains. Nevertheless, expression of human DRK1 channels in Xenopus oocytes showed that mouse, rat and human Kv 2.1 channels have distinct pharmacological and electrophysiological properties. The observed differences in activation, voltage-dependence, 4-aminopyridine sensitivity and single-channel conductance have to be attributed to amino acid substitutions in the amino-and/or carboxy-terminal cytoplasmic domains. Obviously, these domains of Kv 2.1 channels influence biophysical K+ channel properties, which are thought to be determined solely by the membrane spanning core domain of potassium channels.

  19. Identification and analysis of cation channel homologues in human pathogenic fungi.

    Directory of Open Access Journals (Sweden)

    David L Prole

    Full Text Available Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+, calcium (Ca(2+ and transient receptor potential (Trp channels, but not sodium (Na(+ channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+ uniporter (MCU. In contrast to humans, which express many K(+, Ca(2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+, Ca(2+ and Trp channel homologues. Furthermore, the sequences of fungal K(+, Ca(2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

  20. Human beta-defensin 1, a new animal toxin-like blocker of potassium channel.

    Science.gov (United States)

    Feng, Jing; Xie, Zili; Yang, Weishan; Zhao, Yonghui; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Chen, Zongyun; Wu, Yingliang

    2016-04-01

    The discovery of human β-defensin 2 (hBD2), as a Kv1.3 channel inhibitor with the unique molecular mechanism and novel immune modulatory function, suggests that human β-defensins are a novel class of channel ligands. Here, the function and mechanism of the human β-defensin 1 (hBD1) binding to potassium channels was investigated. Based on the structural similarity between hBD1 and Kv1.3 channel-sensitive hBD2, hBD1 was found to selectively inhibit human and mouse Kv1.3 channels with IC50 values of 11.8 ± 3.1 μM and 13.2 ± 4.0 μM, respectively. Different from hBD2 modifying Kv1.3 channel activation and increasing activation time constant, hBD1 did not affect the activation feature of both human and mouse Kv1.3 channels. In comparison with hBD2 simultaneously interacting with the extracellular S1-S2 linker and pore region of Kv1.3 channel, the chimeric channel and mutagenesis experiments showed that hBD1 only bound to the extracellular pore region of Kv1.3 channel instead of extracellular S1-S2 linker or S3-S4 linker. Together, these findings enhance knowledge of hBD1 as a new immune-related Kv1.3 channel blocker and highlight the major functional differences between hBD1 and hBD2 to explore in future research.

  1. Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

    Directory of Open Access Journals (Sweden)

    Berg Ulrike

    2009-04-01

    Full Text Available Abstract Background Granulosa cells (GCs represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (KCa of big conductance (BKCa, which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of KCa channels (including BKCa beta-subunits, and 2. biophysical properties of BKCa channels. Methods GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique. Results We identified two KCa types in human GCs, the intermediate- (IK and the small-conductance KCa (SK. Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by KCa blockers (TRAM-34, apamin. Functional IK channels were also demonstrated by electrophysiological recording of single KCa channels with distinctive features. Both, IK and BKCa channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BKCa channel revealed the presence of mRNAs encoding several BKCa beta-subunits (beta2, beta3, beta4 in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BKCa channels which we observed in electrophysiological recordings. Conclusion Functional and molecular studies indicate the presence of active IK and SK

  2. The sedimentary dynamics in natural and human-influenced delta channel belts

    NARCIS (Netherlands)

    Hobo, N.

    2015-01-01

    This study investigates the increased anthropogenic influence on the within-channel belt sedimentary dynamics in the Rhine delta. To make this investigation, the sedimentary dynamics within the life-cycle of a single channel belt were reconstructed for three key periods of increasing human impact, w

  3. Tetrachromacy of human vision: spectral channels and primary colors

    Science.gov (United States)

    Gavrik, Vitali V.

    2002-06-01

    Full-color imaging requires four channels as, in contrast to a colorimeter, can add no primary to matched scene colors themselves. An ideal imaging channel should have the same spectral sensitivity of scene recording as a retinal receptor and evoke the same primary color sensation. The alternating matching functions of a triad of real primaries are inconsistent with the three cones but explicable of two pairs of independent opponent receptors with their alternating blue-yellow and green-red chromatic axes in the color space. Much other controversy of trichromatic approach can also be explained with the recently proposed intra- receptor processes in the photopic rod and cone, respectively. Each of their four primary sensations, unmixed around 465, 495, 575, and 650 nm, is evoked within a different spectral region. The current trichromatic photographic systems have been found separately to approximate the blue and red receptors, as well as their spectral opponency against the respective yellow and blue- green receptors simulated with a single middle-wave imaging channel. The channel sensitivities are delimited by the neutral points of rod and cone and cannot simulate the necessary overlap of non-opponent channels for properly to render some mixed colors. The yellow and cyan positive dyes closely control the brightness of blue and red sensations, respectively. Those red and blue respectively to control the yellow and blue-green sensations on brightness scales are replaced by magenta dye, controlling them together. Accurate rendering of natural saturation metameric colors, problematic blue-green, purple-red, and low-illumination colors requires to replace the hybrid 'green' channel with the blue-green and yellow channels.

  4. Constitutive activity of the human TRPML2 channel induces cell degeneration.

    Science.gov (United States)

    Lev, Shaya; Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Bach, Gideon; Minke, Baruch

    2010-01-22

    The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca(2+)-permeable and inhibited by low extracytosolic pH but not Ca(2+) regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca(2+) overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation.

  5. Carbon monoxide stimulates the Ca2(+)-activated big conductance k channels in cultured human endothelial cells.

    Science.gov (United States)

    Dong, De-Li; Zhang, Yan; Lin, Dao-Hong; Chen, Jun; Patschan, Susann; Goligorsky, Michael S; Nasjletti, Alberto; Yang, Bao-Feng; Wang, Wen-Hui

    2007-10-01

    We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca(2+)-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N(G)-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N(G)-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

  6. Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.

    Science.gov (United States)

    Hirano-Iwata, Ayumi; Ishinari, Yutaka; Yoshida, Miyu; Araki, Shun; Tadaki, Daisuke; Miyata, Ryusuke; Ishibashi, Kenichi; Yamamoto, Hideaki; Kimura, Yasuo; Niwano, Michio

    2016-05-24

    Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

  7. Transient receptor potential canonical type 3 channels and blood pressure in humans

    DEFF Research Database (Denmark)

    Thilo, Florian; Baumunk, Daniel; Krause, Hans;

    2009-01-01

    There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood...

  8. Statistical characterization of the dynamic human body communication channel at 45 MHz.

    Science.gov (United States)

    Nie, Zedong; Ma, Jingjing; Chen, Hong; Wang, Lei

    2013-01-01

    The dynamic human body communication (HBC) propagation channel at 45 MHz was statistical characterized in this paper. A large amount of measurement data has been gathered in practical environment with real activities -treadmill running at different speeds in a lab room. The received power between two lower legs was acquired from three volunteers, with more than 60,000 snap shot of data in total. The statistical analyses confirmed that the HBC propagation channel at 45 MHz followed the Gamma and Lognormal distributions at the slower (2 km/h and 4 km/h) and faster (6 km/h and 8 km/h) running activities, respectively. The channel is insensitive to body motion with the maximum average fade duration is 0.0413 s and the most averaging bad channel duration time being less than 60 ms with the percentage of the bad channel duration time being less than 4.35%.

  9. The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex.

    Science.gov (United States)

    Syeda, Shameem Sultana; Carlson, Erick J; Miller, Melissa R; Francis, Rawle; Clapham, David E; Lishko, Polina V; Hawkinson, Jon E; Hook, Derek; Georg, Gunda I

    2016-02-19

    The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

  10. Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation.

    Science.gov (United States)

    Feng, Jing; Yang, Weishan; Xie, Zili; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Hu, Hongzhen; Chen, Zongyun; Wu, Yingliang

    2015-06-19

    Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

  11. Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel.

    Science.gov (United States)

    Rojas, C V; Neely, A; Velasco-Loyden, G; Palma, V; Kukuljan, M

    1999-01-01

    Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

  12. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    OpenAIRE

    Soldatov, N M

    1992-01-01

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of thes...

  13. 心功能不同的风湿性心脏瓣膜病心房颤动患者超极化激活环核苷酸门控阳离子通道4的表达水平研究%Expression Level of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 4 in Patients With Atrial Fibrillation Associated With Rheumatic Valvular Heart Disease of Different Cardiac Functions

    Institute of Scientific and Technical Information of China (English)

    张健; 李发鹏; 甘天翊; 许国军; 何卫; 周贤惠; 汤宝鹏; 李耀东; 郭霞

    2015-01-01

    目的:探讨超极化激活环核苷酸门控阳离子通道4(HCN4)基因在风湿性心脏瓣膜病心房颤动伴心力衰竭患者与心功能正常的风湿性心脏瓣膜病心房颤动患者心房肌中的表达水平。方法选取2008—2011年新疆医科大学第一附属医院因心脏瓣膜病需接受开胸换瓣手术患者45例,根据其心功能分级,将美国纽约心脏病协会( NYHA)分级为Ⅱ~Ⅲ级者27例作为试验组,将心功能正常者18例作为对照组。采用实时荧光定量PCR( Real-time PCR)和蛋白质免疫印迹法( Western-blotting)分别测定两组患者HCN4 mRNA及蛋白表达水平。结果对照组HCN4 mRNA表达水平为(1.12±0.69),低于试验组的(4.91±1.51)(t =0.021,P <0.05)。对照组 HCN4蛋白表达水平为(1.02±0.15),低于试验组的(2.01±0.92)(t=0.031,P<0.001)。结论 HCN4在心力衰竭与心功能正常的风湿性心脏瓣膜病心房颤动患者的心房肌中均有表达,且随着心功能不全的加重,HCN4表达水平上调。%Objective To investigate the expression level of hyperpolarization -activated cyclic nucleotide -gated channel 4(HCN4)in the atrial muscle of patients with atrial fibrillation associated with rheumatic valvular heart disease with heart failure or normal cardiac function. Methods We enrolled 45 patients with valvular heart disease who were going to receive thoracotomy for valve replacement in the First Affiliated Hospital of Xinjiang medical University from 2008 to 2011. According to cardiac functional grading,we assigned 27 patients who were at grade Ⅱ-Ⅲin NYHA grading into trial group and assigned 18 patients with normal cardiac function as control group. Real -time PCR and Western -blotting were employed to determine mRNA level and protein expression level of HCN4 of the two groups. Results The mRNA expression level of HCN4 in control group was(1. 12 ±0. 69),lower than that of trial group which was(4

  14. Communication channel modeling of human forearm with muscle fiber tissue characteristics.

    Science.gov (United States)

    Zhang, Shuang; Pun, Sio Hang; Mak, Peng Un; Qin, Yu-Ping; Liu, Yi-He; Vai, Mang I

    2016-09-14

    Human-Body Communication (HBC) is a wireless communication method using the human body tissue as a transmission medium for signals. This paper on the basis of human muscle fiber tissues' characteristics, it is first proposed to establish the analytical model of galvanic coupling human-body communication channel. In this model, the parallel and the transverse electrical characteristics of muscular tissue are fully considered, and the model accurately presents the transmission mechanism of galvanic coupling human-body communication signals in the channel. At last, through compare with the experimental results and calculation results, the maximum error of the model is 22.4% and the average error is 14.2% within the frequency range.

  15. Ion channels in human red blood cell membrane: actors or relics?

    Science.gov (United States)

    Thomas, Serge L Y; Bouyer, Guillaume; Cueff, Anne; Egée, Stéphane; Glogowska, Edyta; Ollivaux, Céline

    2011-04-15

    During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease.

  16. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Directory of Open Access Journals (Sweden)

    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  17. Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

    Science.gov (United States)

    Gąsiorowska, Justyna; Teisseyre, Andrzej; Uryga, Anna; Michalak, Krystyna

    2015-08-01

    Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 μM for xanthohumol and about 7.8 μM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 μM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents' activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

  18. The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence

    DEFF Research Database (Denmark)

    Bennekou, P.; Barksmann, T. L.; Christophersen, P.

    2006-01-01

    The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte...

  19. Anion conductance of the human red cell is carried by a maxi-anion channel

    DEFF Research Database (Denmark)

    Glogowska, Edyta; Dyrda, Agnieszka; Cueff, Anne

    2010-01-01

    played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance...... channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating...... that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator...

  20. Effects of large conductance Ca(2+)-activated K(+) channels on nitroglycerin-mediated vasorelaxation in humans

    DEFF Research Database (Denmark)

    Gruhn, Nicolai; Boesgaard, Søren; Eiberg, Jonas

    2002-01-01

    Nitric oxide (NO)-induced vasorelaxation and the regulation of endothelial superoxide anion levels is partly mediated by vascular large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Nitroglycerin acts through the release of NO and its effect is modulated by changes in endothelial superoxide...... levels. This study examines the effect of BK(Ca) channel blockade on nitroglycerin-induced vasorelaxation in human arterial and venous vascular segments and whether responses to BK(Ca) channel blockade are influenced by the development of venous nitroglycerin tolerance. Dose-relaxation curves...... suggest that primarily arterial effects of nitroglycerin are significantly inhibited by changes in the activity of the endothelial BK(Ca) channels. Although endothelial BK(Ca) are likely regulators of mechanisms underlying arterial tolerance development to nitroglycerin, they do not appear to play a role...

  1. Electrogenic transport and K(+) ion channel expression by the human endolymphatic sac epithelium.

    Science.gov (United States)

    Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J; Jung, JinSei; Moon, In Seok; Moon, Sung K; Choi, Jae Young

    2015-12-14

    The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid.

  2. Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents.

    Science.gov (United States)

    Yu, Jing; Park, Mi-Hyeong; Jo, Su-Hyun

    2015-01-05

    Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2±4.2μM and 33.4±3.2μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from -10mV to +30mV, while hydrocortisone׳s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel׳s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently, and that Kv1.5 channels were more sensitive to hydrocortisone than to cortisone. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Na+-permeable channels of human sperm membrane re- assembled into giant liposome

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Previous data showed that a Na+-transmembrane flux was accompanied with acrosome reaction of sperm. However, the electrophysiological recording and characterization of Na+ current in human sperm membrane have not been yet reported. In the present investigation, membrane proteins extracted from human sperms were reassembled into liposome bilayer, and then the liposomes were fused by dehydration-rehydration into giant liposomes with the diameter of more than 10 mm. By patch clamping the giant liposomes two kinds of single channel currents were recorded in a NaCl solution system. Both of them were Na+-carried, TTX-sensitive and strongly rectifying, but with different unit conductance and open probability. Moreover, bursting activity and channel-substates as well as two open time constants were observed in the larger channel.

  4. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

  5. The voltage-gated sodium channel nav1.8 is expressed in human sperm.

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

    Full Text Available The role of Na(+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM, the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+-containing or Ca(2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+, [Na(+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

  6. Distribution and function of sodium channel subtypes in human atrial myocardium

    NARCIS (Netherlands)

    Kaufmann, Susann G.; Westenbroek, Ruth E.; Maass, Alexander H.; Lange, Volkmar; Renner, Andre; Wischmeyer, Erhard; Bonz, Andreas; Muck, Jenny; Ertl, Georg; Catterall, William A.; Scheuer, Todd; Maier, Sebastian K. G.

    2013-01-01

    Voltage-gated sodium channels composed of a pore-forming alpha subunit and auxiliary beta subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohist

  7. Functional expression of voltage-gated calcium channels in human melanoma.

    Science.gov (United States)

    Das, A; Pushparaj, C; Bahí, N; Sorolla, A; Herreros, J; Pamplona, R; Vilella, R; Matias-Guiu, X; Martí, R M; Cantí, C

    2012-03-01

    The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression. © 2012 John Wiley & Sons A/S.

  8. T-type calcium channel expression in cultured human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Xianjie Wen; Shiyuan Xu; Lingling Wang; Hua Liang; Chengxiang Yang; Hanbing Wang; Hongzhen Liu

    2011-01-01

    Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions. Previous studies have revealed Land N-type calcium channels in SH-SY5Y cells. However, the distribution of the low -voltage activated calcium channel (namely called T-type calcium channel, including Cav3.1, Cav3.2, and Cav3.3) in SH-SY5Y cells remains poorly understood. The present study detected mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2, and Cav3.3) in cultured SH-SY5Y cells using real-time polymerase chain reaction (PCR) and western blot analysis. Results revealed mRNA and protein expression from all three T-type calcium channel subtypes in SH-SY5Y cells. Moreover,Cav3.1 was the predominant T-type calcium channel subtype in SH-SY5Y cells.

  9. Comparison of the channelized Hotelling and human observers for lesion detection in hepatic SPECT imaging

    Science.gov (United States)

    King, Michael A.; de Vries, Daniel J.; Soares, Edward J.

    1997-04-01

    The relative rankings of the channelized Hotelling model observer were compared to those of the human observers for the task of detecting 'hot' tumors in simulated hepatic SPECT slices. The signal-to-noise ratios (SNRs) were determined using eighty images for each of three slice locations. The acquisition and processing strategies investigated were: (1) imaging solely primary photons, (2) imaging primary plus scatter within a 20% symmetric energy window for Tc-99m, (3) imaging with primary plus an elevated amount of scatter, (4) energy-spectrum-based scatter compensation of the primary plus scatter acquisitions, and (5) energy-spectrum-based scatter compensation of the acquisitions with an elevated amount of scatter. Both square non-overlapping channels (SQR), and overlapping difference- of-Gaussian channels (DOG) were incorporated into the Hotelling model observer. When the scatter compensation results were excluded, both channelized Hotelling model observers exhibited a strong correlation with the rankings of the human-observers. With the inclusion of the scatter compensation results, only with the DOG model observer was the null-hypothesis of no correlation rejected at the p equals 0.05 level. It is concluded that further investigation of the channel model used with the Hotelling observer is indicated to determine if better correlation can be obtained.

  10. Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

    Science.gov (United States)

    Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

    2017-06-01

    Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

    Science.gov (United States)

    Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

    2011-08-15

    Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation.

    Science.gov (United States)

    Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

    2010-06-01

    Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

  13. Channel Modeling of Human Somatosensory Nanonetwork: Body Discriminative Touch and Proprioception Perspective

    Directory of Open Access Journals (Sweden)

    Partha Pratim Ray

    2013-10-01

    Full Text Available Nanonetwork design and analysis has become a very interesting topic in recent years. Though this area of research is in its formative stage, it definitely posses a strong integrity in finding out numerous applications in medical and allied sciences. Nanonetworking is indeed a nature built foundation which comprises human intra body communications. Somatosensory system is the one of the critical and must have systems of human body. This literature concentrates on the body discriminative touch and proprioception mechanism of somatosensory system. This particular system is well architecture by medial lemniscal pathway, in human body for transduction of touch and proprioceptive information. This paper seeks out the novel communication channel model of somatosensory system. The working principle of the channel model is established by an equivalent Moore machine. A novel algorithm MLP is proposed after its name, medial lemniscal pathway. A novel naomachine and appropriate processing unit are also devised, based on the automaton.

  14. Functional Importance of L- and P/Q-Type Voltage-Gated Calcium Channels in Human Renal Vasculature

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Poulsen, Christian B; Walter, Steen

    2011-01-01

    in kidney function. It was hypothesized that human renal vascular excitation-contraction coupling involves different subtypes of channels. In human renal artery and dissected intrarenal blood vessels from nephrectomies, PCR analysis showed expression of L-type (Ca(v) 1.2), P/Q-type (Ca(v) 2.1), and T-type......, and L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers......Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved...

  15. [Study on the effect of Klotho gene interferred by plasmid-mediated short hairpin RNA (shRNA) on sinoatrial node pacing channel gene].

    Science.gov (United States)

    Cai, Yingying; Wang, Han; Hou, Yanbin; Fang, Chenli; Tian, Peng; Wang, Guihua; Li, Lu; Deng, Juelin

    2013-06-01

    The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  16. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    Science.gov (United States)

    Soldatov, N M

    1992-05-15

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of these sites correlate with RNA splice sites, indicating that the molecular diversity of the transcripts is a result of alternative splicing. The fourth diversity region is located at the C-terminal region and comprises insertions and deletions. It is suggested that these variations may give rise to multiple subforms of HFCC with altered electrophysiological properties.

  17. Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells.

    Science.gov (United States)

    Morachevskaya, Elena; Sudarikova, Anastasiya; Negulyaev, Yuri

    2007-04-01

    This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.

  18. Otilonium bromide inhibits calcium entry through L-type calcium channels in human intestinal smooth muscle.

    Science.gov (United States)

    Strege, P R; Evangelista, S; Lyford, G L; Sarr, M G; Farrugia, G

    2004-04-01

    Otilonium bromide (OB) is used as an intestinal antispasmodic. The mechanism of action of OB is not completely understood. As Ca(2+) entry into intestinal smooth muscle is required to trigger contractile activity, our hypothesis was that OB blocked Ca(2+) entry through L-type Ca(2+) channels. Our aim was to determine the effects of OB on Ca(2+), Na(+) and K(+) ion channels in human jejunal circular smooth muscle cells and on L-type Ca(2+) channels expressed heterologously in HEK293 cells. Whole cell currents were recorded using standard patch clamp techniques. Otilonium bromide (0.09-9 micromol L(-1)) was used as this reproduced clinical intracellular concentrations. In human circular smooth muscle cells, OB inhibited L-type Ca(2+) current by 25% at 0.9 micromol L(-1) and 90% at 9 micromol L(-1). Otilonium bromide had no effect on Na(+) or K(+) currents. In HEK293 cells, 1 micromol L(-1) OB significantly inhibited the expressed L-type Ca(2+) channels. Truncation of the alpha(1C) subunit C and N termini did not block the inhibitory effects of OB. Otilonium bromide inhibited Ca(2+) entry through L-type Ca(2+) at concentrations similar to intestinal tissue levels. This effect may underlie the observed muscle relaxant effects of the drug.

  19. Urban river restoration: implications on channel sedimentation patterns and associated ecosystem and human health

    Science.gov (United States)

    Gibbs, H.; Gurnell, A.; Heppell, K.; Spencer, K.

    2012-04-01

    Urban river restoration, which alters the physical and hydraulic conditions of rivers, creates rivers favourable to increased sedimentation through greater sediment availability and heterogeneous flow patterns. Sediments, particularly finer-grained, store contaminants including metals which can have detrimental impacts upon aquatic ecosystems and potentially human health. This research therefore looks at the effect of urban river restoration practices upon sedimentation patterns, the associated changes in sediment metal storage and the potential impact upon river function and use in terms of the aquatic ecosystem and human health. Research was undertaken at four sites on urban rivers in London. The spatial extent of different bed sediment types (unvegetated gravel, sand, finer and sediment around in-channel vegetation) in adjacent restored and unrestored stretches was mapped in July 2010. Additionally, sediments were sampled through the year and analysed for a range of metals and sediment characteristics. Two sites (Chinbrook Meadows and Sutcliffe Park) showed a clear difference in bed sediment type channel cover between the restored and unrestored stretches. The majority of the concrete-lined unrestored stretch at Chinbrook Meadows had no sediment deposition, whereas the restored stretch had over half of the channel occupied by finer sediment either on the open channel bed or accumulated around in-channel vegetation. At Sutcliffe Park, the dominant bed sediment type in the restored stretch was finer sediment on the open bed and accumulated around in-channel vegetation, whereas in the unrestored stretch the dominant bed sediment type was gravel. At both sites there were significant differences in metal concentrations and sediment characteristics between bed sediment types. Metal concentrations, organic matter and % <63µm were generally higher in the finer sediment whether on the open bed or around in-channel vegetation. Total loadings of all metals were greater in

  20. Purinergic regulation of CFTR and Ca2+ -activated Cl- channels and K+ channels in human pancreatic duct epithelium

    DEFF Research Database (Denmark)

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-01-01

    dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have...... mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3...... receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct...

  1. Geometry of human vocal folds and glottal channel for mathematical and biomechanical modeling of voice production.

    Science.gov (United States)

    Sidlof, Petr; Svec, Jan G; Horácek, Jaromír; Veselý, Jan; Klepácek, Ivo; Havlík, Radan

    2008-01-01

    Current models of the vocal folds derive their shape from approximate information rather than from exactly measured data. The objective of this study was to obtain detailed measurements on the geometry of human vocal folds and the glottal channel in phonatory position. A non-destructive casting methodology was developed to capture the vocal fold shape from excised human larynges on both medial and superior surfaces. Two female larynges, each in two different phonatory configurations corresponding to low and high fundamental frequency of the vocal fold vibrations, were measured. A coordinate measuring machine was used to digitize the casts yielding 3D computer models of the vocal fold shape. The coronal sections were located in the models, extracted and fitted by piecewise-defined cubic functions allowing a mathematical expression of the 2D shape of the glottal channel. Left-right differences between the cross-sectional shapes of the vocal folds were found in both the larynges.

  2. Pharmacological Investigation of Voltage-dependent Ca2+ Channels in Human Ejaculatory Sperm in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Lu; LIU Jihong; LI Jiagui; YE Zhangqun

    2006-01-01

    The types of the voltage-dependent calcium channels (VDCCs) in human ejaculatory sperm and the effects of calcium channel blocker (CCB) on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine (NIF, inhibitor of L-type VDCC) and ω-conotoxin (GVIA, inhibitor of N-type VDCC) were compared and analyzed statistically. The results showed that NIF (1, 5, 10 μmol/L)could not only significantly affect human sperm's shape but also spermatozoa motility after incubated at least 10 min in vitro (P<0.001). GVIA (0.1, 0.5 and 1 μmol/L) could just only significantly affect human sperm's progressive motility (a %+b %) after incubated for 20 min in vitro (P<0.01), but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

  3. Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization.

    Science.gov (United States)

    Mazzo, Francesca; Zwart, Ruud; Serratto, Giulia Maia; Gardinier, Kevin M; Porter, Warren; Reel, Jon; Maraula, Giovanna; Sher, Emanuele

    2016-08-01

    Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that

  4. Polyunsaturated fatty acids are potent openers of human M-channels expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Liin, Sara I; Karlsson, Urban; Bentzen, Bo Hjorth

    2016-01-01

    , by shifting the conductance-versus-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μM). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel. CONCLUSIONS: These findings suggest that circulating...... acids reduce neuronal excitability. This article is protected by copyright. All rights reserved....

  5. Measurement and analysis of channel attenuation characteristics for an implantable galvanic coupling human-body communication.

    Science.gov (United States)

    Zhang, Shuang; Pun, Sio Hang; Mak, Peng Un; Qin, Yu-Ping; Liu, Yi-He; Vai, Mang I

    2016-11-14

    In this study, an experiment was designed to verify the low power consumption of galvanic coupling human-body communication. A silver electrode (silver content: 99%) is placed in a pig leg and a sine wave signal with the power of 0 dBm is input. Compared with radio frequency communication and antenna transmission communication, attenuation is reduced by approximately 10 to 15 dB, so channel characteristics are highly improved.

  6. Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

    Science.gov (United States)

    Crottès, David; Félix, Romain; Meley, Daniel; Chadet, Stéphanie; Herr, Florence; Audiger, Cindy; Soriani, Olivier; Vandier, Christophe; Roger, Sébastien; Angoulvant, Denis; Velge-Roussel, Florence

    2016-04-01

    Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

  7. Effects of environmental changes and human impact on the functioning of mountain river channels, Carpathians, southern Poland

    Directory of Open Access Journals (Sweden)

    Krzemień Kazimierz

    2015-09-01

    Full Text Available In the northern slope of the Carpathian Mountains and in their foreland, river and stream channels have been significantly transformed by human impact. These transformations result from changing land use in river basins and direct interference with river channels (alluvia extraction, engineering infrastructure, channel straightening. Anthropogenic impacts cause significant changes in the channel system patterns leading to increased impact of erosion. This mainly leads to the channelling of the fluvial system. This article reviews studies of structure and dynamics of Carpathian river channels conducted based on the methodology of collection of data on channel systems, developed in the Department of Geomorphology of the Institute of Geography and Spatial Management, Jagiellonian University.

  8. Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels.

    Science.gov (United States)

    Ruchala, Iwona; Cabra, Vanessa; Solis, Ernesto; Glennon, Richard A; De Felice, Louis J; Eltit, Jose M

    2014-07-01

    Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells.

  9. Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

    Energy Technology Data Exchange (ETDEWEB)

    Bevilacqua, M.; Vago, T.; Norbiato, G. (Servizio di Endocrinologia, Milano, (Italy))

    1991-02-01

    Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine ({sup 3}H)-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca{sup 2}{sup +} and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca{sup 2}{sup +}-containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37{degree}C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37{degree}C decreased the affinity of the binding; this effect was counteracted by the addition of Ca{sup 2}{sup +} to the medium. This result was consistent with a competition between Ca{sup 2}{sup +} and PC. The effect of PC incubation at 4{degree}C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca{sup 2}{sup +}.

  10. Evaluation of six channelized Hotelling observers in combination with a contrast sensitivity function to predict human observer performance

    Science.gov (United States)

    Goffi, Marco; Veldkamp, Wouter J. H.; van Engen, Ruben E.; Bouwman, Ramona W.

    2015-03-01

    Standard methods to quantify image quality (IQ) may not be adequate for clinical images since they depend on uniform backgrounds and linearity. Statistical model observers are not restricted to these limitations and might be suitable for IQ evaluation of clinical images. One of these statistical model observers is the channelized Hotelling observer (CHO), where the images are filtered by a set of channels. The aim of this study was to evaluate six different channel sets, with an additional filter to simulate the human contrast sensitivity function (CSF), in their ability to predict human observer performance. For this evaluation a two alternative forced choice experiment was performed with two types of background structures (white noise (WN) and clustered lumpy background (CLB)), 5 disk-shaped objects with different diameters and 3 different signal energies. The results show that the correlation between human and model observers have a diameter dependency for some channel sets in combination with CLBs. The addition of the CSF reduces this diameter dependency and in some cases improves the correlation coefficient between human- and model observer. For the CLB the Partial Least Squares channel set shows the highest correlation with the human observer (r2=0.71) and for WN backgrounds it was the Gabor-channel set with CSF (r2=0.72). This study showed that for some channels there is a high correlation between human and model observer, which suggests that the CHO has potential as a tool for IQ analysis of digital mammography systems.

  11. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

    Full Text Available Bisphenol A (BPA has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.

  12. Expression and pharmacology of endogenous Cav channels in SH-SY5Y human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Silmara R Sousa

    Full Text Available SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1-3, β1, β3, β4, γ1, γ4-5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.

  13. High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes

    DEFF Research Database (Denmark)

    Thilo, Florian; Lee, Marlene; Xia, Shengqiang

    2014-01-01

    Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxid...

  14. A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics

    NARCIS (Netherlands)

    Groenewegen, WA; Bezzina, CR; van Tintelen, JP; Hoorntje, TM; Mannens, MMAM; Wilde, AAM; Jongsma, HJ; Rook, MB

    2003-01-01

    The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. Objective: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. Methods: The human cardiac sodium channel gene SCN5A was s

  15. Human aquaporin-11 is a water and glycerol channel and localizes in the vicinity of lipid droplets in human adipocytes.

    Science.gov (United States)

    Madeira, Ana; Fernández-Veledo, Sonia; Camps, Marta; Zorzano, Antonio; Moura, Teresa F; Ceperuelo-Mallafré, Victoria; Vendrell, Joan; Soveral, Graça

    2014-09-01

    For a long time Aquaporin-7 has been the only aquaporin associated with the adipose tissue, and its dysregulation has been linked to the underlying mechanisms of obesity. However, the presence of alternative glycerol channels within the adipose tissue has been postulated, which has prompted us to the search of alternate glycerol transport routes in adipocytes. In view of this, it is hypothesized that Aquaporin-11 (AQP11) would have a role in adipocyte cell biology. The expression, the localization and the function of human AQP11 (hAQP11) in cultured differentiated adipocytes were investigated. Gene expression analysis revealed the presence of AQP11 in both subcutaneous and visceral human mature adipocytes. It is found that hAQP11 is primarily located intracellularly in human adipocytes and partially colocalizes with perilipin, pointing towards AQP11 preferential location in the vicinity of lipid droplets. Overexpression of hAQP11 in 3T3-L1 adipocytes enabled to validate its function as a water channel and reveal its glycerol permeation activity. hAQP11 permeates both water and glycerol, localizing in the vicinity of lipid droplets in human adipocytes. © 2014 The Obesity Society.

  16. Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

    Directory of Open Access Journals (Sweden)

    Rossie Sandra

    2004-06-01

    Full Text Available Abstract Background Intermediate-conductance, calcium-activated potassium channels (IKs modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1 expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D. Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is

  17. Deltamethrin Inhibits the Human T-type Voltage-Sensitive Calcium Channel (Cav3.2

    Directory of Open Access Journals (Sweden)

    Steven B. Symington

    2009-01-01

    Full Text Available The goal of this study was to determine the effect of deltamethrin, a pyrethroid insecticide, on CaV3.2, a human T-type voltage-sensitive calcium channel expressed in Xenopus laevis (X.laevis oocytes. Cav3.2 cDNA was transcribed into cRNA; the cRNA was then injected into X.laevis oocytes and electrophysiologically characterized using the two-electrode voltage clamp technique with Ba2+ as a charge carrier. Deltamethrin (10-7 M reduced peak current in a nonreversible manner compared to the untreated control, but had no effect on the voltagedependent activation and inactivation kinetics. These findings confirm that human CaV3.2 is a target for deltamethrin and quite possibly other pyrethroid insecticides. These studies provide insight into the molecular mechanisms of the effect that pyrethroids have on voltage-sensitive calcium channels in general. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced toxicity and expand our knowledge of the structure-activity relationships of pyrethroids with regard to their action on voltage-sensitive calcium channels.

  18. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel

    OpenAIRE

    Liu, Zhenzhen; Jiang, Tianyu; Wang, Beilei; Ke, Bowen; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-01-01

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, th...

  19. Acacetin Blocks Kv1.3 Channels and Inhibits Human T Cell Activation

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    2014-10-01

    Full Text Available Backgrounds/Aims: Acacetin, a natural flavonoid compound, has been proven to exert anti-inflammatory and immunomodulatory effects. Kv1.3 channels, highly expressed in human T cells, are attractive therapeutic targets to treat inflammatory and immunological disorders. The present study was designed to characterize the inhibition of Kv1.3 channels by Acacetin in human T cells and examine its role in T cell activation. Methods: Whole-cell patch-clamp was applied to record the Kv1.3 and KCa currents in human T cells; Western blot was used to detect Kv1.3 expression as well as NFAT1 and NF-κB activity; Fluo-4, CCK-8 and an ELISA kit were used to measure Ca2+ influx, proliferation, and IL-2 secretion, respectively. Results: Acacetin decreased the Kv1.3 current, accelerated the decay rate and negatively shifted the steady-state inactivation curves in a concentration-dependent manner. The IC50 values at +40 mV for peak and the current at end of pulse were 21.09 ± 2.75 and 3.63 ± 0.25 µmol/L, respectively. Treatment with Acacetin for 24 h significantly inhibited Kv1.3 protein expression. Additionally, paralleling Kv1.3 inhibition, Acacetin also inhibited Ca2+ influx, the Ca2+-activated transcription factors NFAT1, NF-κB p65/p50 activity, and proliferation as well as IL-2 production. Small interfering RNA against Kv1.3 reduced the inhibitory effect of Acacetin on IL-2 secretion. Conclusions: Acacetin blocks the Kv1.3 channel and inhibits human T cell activation. This action most likely contributes to its immunomodulatory and anti-inflammatory actions.

  20. Transmural expression of ion channels and transporters in human nondiseased and end-stage failing hearts

    DEFF Research Database (Denmark)

    Soltysinska, Ewa; Olesen, Søren-Peter; Christ, Torsten

    2009-01-01

    The cardiac action potential is primarily shaped by the orchestrated function of several different types of ion channels and transporters. One of the regional differences believed to play a major role in the progression and stability of the action potential is the transmural gradient of electrical...... activity across the ventricular wall. An altered balance in the ionic currents across the free wall is assumed to be a substrate for arrhythmia. A large fraction of patients with heart failure experience ventricular arrhythmia. However, the underlying substrate of these functional changes is not well......-established as expression analyses of human heart failure (HF) are sparse. We have investigated steady-state RNA levels by quantitative polymerase chain reaction of ion channels, transporters, connexin 43, and miR-1 in 11 end-stage HF and seven nonfailing (NF) hearts. The quantifications were performed on endo-, mid...

  1. Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

    Science.gov (United States)

    Partiseti, M; Choquet, D; Diu, A; Korn, H

    1992-06-01

    The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.

  2. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel.

    Science.gov (United States)

    Lishko, Polina V; Botchkina, Inna L; Fedorenko, Andriy; Kirichok, Yuriy

    2010-02-05

    Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.

  3. TRPV channel expression in human skin and possible role in thermally induced cell death.

    Science.gov (United States)

    Radtke, Christine; Sinis, Nektarios; Sauter, Michael; Jahn, Sabrina; Kraushaar, Udo; Guenther, Elke; Rodemann, H Peter; Rennekampff, Hans-Oliver

    2011-01-01

    Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.

  4. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  5. Development of membrane ion channels during neural differentiation from human embryonic stem cells.

    Science.gov (United States)

    Mirsadeghi, Sara; Shahbazi, Ebrahim; Hemmesi, Katayoun; Nemati, Shiva; Baharvand, Hossein; Mirnajafi-Zadeh, Javad; Kiani, Sahar

    2017-09-09

    For human embryonic stem cells (hESCs) to differentiate into neurons, enormous changes has to occur leading to trigger action potential and neurotransmitter release. We attempt to determine the changes in expression of voltage gated channels (VGCs) and their electrophysiological properties during neural differentiation. The relative expressions of α-subunit of voltage gated potassium, sodium and calcium channels were characterized by qRT-PCR technique. Patch clamp recording was performed to characterize the electrophysiological properties of hESCs during their differentiation into neuron-like cells. Relative expression of α-subunit of channels changed significantly. 4-AP and TEA sensitive outward currents were observed in all stages, although TEA sensitive currents were recorded once in rosette structure. Nifedipine and QX314 sensitive inward currents were recorded only in neuron-like cells. K(+) currents were recorded in hESCs and rosette structure cells. Inward currents, sensitive to Nifedipine and QX314, were recorded in neuron-like cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Mapping ECoG channel contributions to trajectory and muscle activity prediction in human sensorimotor cortex

    Science.gov (United States)

    Nakanishi, Yasuhiko; Yanagisawa, Takufumi; Shin, Duk; Kambara, Hiroyuki; Yoshimura, Natsue; Tanaka, Masataka; Fukuma, Ryohei; Kishima, Haruhiko; Hirata, Masayuki; Koike, Yasuharu

    2017-01-01

    Studies on brain-machine interface techniques have shown that electrocorticography (ECoG) is an effective modality for predicting limb trajectories and muscle activity in humans. Motor control studies have also identified distributions of “extrinsic-like” and “intrinsic-like” neurons in the premotor (PM) and primary motor (M1) cortices. Here, we investigated whether trajectories and muscle activity predicted from ECoG were obtained based on signals derived from extrinsic-like or intrinsic-like neurons. Three participants carried objects of three different masses along the same counterclockwise path on a table. Trajectories of the object and upper arm muscle activity were predicted using a sparse linear regression. Weight matrices for the predictors were then compared to determine if the ECoG channels contributed more information about trajectory or muscle activity. We found that channels over both PM and M1 contributed highly to trajectory prediction, while a channel over M1 was the highest contributor for muscle activity prediction. PMID:28361947

  7. Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

    Science.gov (United States)

    Silva, L F G; Nascimento, N R F; Fonteles, M C; de Nucci, G; Moraes, M E; Vasconcelos, P R L; Moraes, M O

    2005-01-01

    To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; Pphentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; Pphentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; Pphentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

  8. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

  9. An optofluidic channel model for in vivo nanosensor networks in human blood

    Science.gov (United States)

    Johari, Pedram; Jornet, Josep M.

    2017-05-01

    In vivo Wireless Nanosensor Networks (iWNSNs) consist of nano-sized communicating devices with unprece- dented sensing and actuation capabilities, which are able to operate inside the human body. iWNSNs are a disruptive technology that enables the monitoring and control of biological processes at the cellular and sub- cellular levels. Compared to ex vivo measurements, which are conducted on samples extracted from the human body, iWNSNs can track (sub) cellular processes when and where they occur. Major progress in the field of na- noelectronics, nanophotonics and wireless communication is enabling the interconnection of nanosensors. Among others, plasmonic nanolasers with sub-micrometric footprint, plasmonic nano-antennas able to confine light in nanometric structures, and single-photon detectors with unrivaled sensitivity, enable the communication among implanted nanosensors in the near infrared and optical transmission windows. Motivated by these results, in this paper, an optofluidic channel model is developed to investigate the communication properties and temporal dynamics between a pair of in vivo nanosensors in the human blood. The developed model builds upon the authors' recent work on light propagation modeling through multi-layered single cells and cell assemblies and takes into account the geometric, electromagnetic and microfluidic properties of red blood cells in the human circulatory system. The proposed model guides the development of practical communication strategies among nanosensors, and paves the way through new nano-biosensing strategies able to identify diseases by detecting the slight changes in the channel impulse response, caused by either the change in shape of the blood cells or the presence of pathogens.

  10. Temporal and spatial mouse brain expression of cereblon, an ionic channel regulator involved in human intelligence.

    Science.gov (United States)

    Higgins, Joseph J; Tal, Adit L; Sun, Xiaowei; Hauck, Stefanie C R; Hao, Jin; Kosofosky, Barry E; Rajadhyaksha, Anjali M

    2010-03-01

    A mild form of autosomal recessive, nonsyndromal intellectual disability (ARNSID) in humans is caused by a homozygous nonsense mutation in the cereblon gene (mutCRBN). Rodent crbn protein binds to the intracellular C-terminus of the large conductance Ca(2+)-activated K(+)channel (BK(Ca)). An mRNA variant (human SITE 2 INSERT or mouse strex) of the BK(Ca) gene (KCNMA1) that is normally expressed during embryonic development is aberrantly expressed in mutCRBN human lymphoblastoid cell lines (LCLs) as compared to wild-type (wt) LCLs. The present study analyzes the temporal and spatial distribution of crbn and kcnma1 mRNAs in the mouse brain by the quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR). The spatial expression pattern of endogenous and exogenous crbn proteins is characterized by immunostaining. The results show that neocortical (CTX) crbn and kcnma1 mRNA expression increases from embryonic stages to adulthood. The strex mRNA variant is >3.5-fold higher in embryos and decreases rapidly postnatally. Mouse crbn mRNA is abundant in the cerebellum (CRBM), with less expression in the CTX, hippocampus (HC), and striatum (Str) in adult mice. The intracytoplasmic distribution of endogenous crbn protein in the mouse CRBM, CTX, HC, and Str is similar to the immunostaining pattern described previously for the BK(Ca) channel. Exogenous hemagglutinin (HA) epitope-tagged human wt- and mutCRBN proteins using cDNA transfection in HEK293T cell lines showed the same intracellular expression distribution as endogenous mouse crbn protein. The results suggest that mutCRBN may cause ARNSID by disrupting the developmental regulation of BK(Ca) in brain regions that are critical for memory and learning.

  11. The effect of pH and ion channel modulators on human placental arteries.

    Directory of Open Access Journals (Sweden)

    Tayyba Y Ali

    Full Text Available Chorionic plate arteries (CPA are located at the maternofetal interface where they are able to respond to local metabolic changes. Unlike many other types of vasculature, the placenta lacks nervous control and requires autoregulation for controlling blood flow. The placental circulation, which is of low-resistance, may become hypoxic easily leading to fetal acidosis and fetal distress however the role of the ion channels in these circumstances is not well-understood. Active potassium channel conductances that are subject to local physicochemical modulation may serve as pathways through which such signals are transduced. The aim of this study was to investigate the modulation of CPA by pH and the channels implicated in these responses using wire myography. CPA were isolated from healthy placentae and pre-contracted with U46619 before testing the effects of extracellular pH using 1 M lactic acid over the pH range 7.4-6.4 in the presence of a variety of ion channel modulators. A change from pH 7.4 to 7.2 produced a 29±3% (n = 9 relaxation of CPA which increased to 61±4% at the lowest pH of 6.4. In vessels isolated from placentae of women with pre-eclampsia (n = 6, pH responses were attenuated. L-methionine increased the relaxation to 67±7% (n = 6; p<0.001 at pH 6.4. Similarly the TASK 1/3 blocker zinc chloride (1 mM gave a maximum relaxation of 72±5% (n = 8; p<0.01 which compared with the relaxation produced by the TREK-1 opener riluzole (75±5%; n = 6. Several other modulators induced no significant changes in vascular responses. Our study confirmed expression of several ion channel subtypes in CPA with our results indicating that extracellular pH within the physiological range has an important role in controlling vasodilatation in the human term placenta.

  12. Activation of stretch-activated channels and maxi-K+ channels by membrane stress of human lamina cribrosa cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2009-01-01

    The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca(2+)](i)).

  13. Block of Human Cardiac Sodium Channels by Lacosamide: Evidence for Slow Drug Binding along the Activation Pathway

    OpenAIRE

    Wang, Ging Kuo; Wang, Sho-Ya

    2014-01-01

    Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na+ channels for its therapeutic action. Cardiac Na+ channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na+ channels. Lacosamide showed little effect on hNav1.5 Na+ currents at 300 µM when cells were held at −140 mV. With 30-second condi...

  14. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action.

    Science.gov (United States)

    Vonderlin, Nadine; Fischer, Fathima; Zitron, Edgar; Seyler, Claudia; Scherer, Daniel; Thomas, Dierk; Katus, Hugo A; Scholz, Eberhard P

    2015-01-01

    Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam.

  15. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

    Science.gov (United States)

    Zsembery, Akos; Boyce, Amanda T; Liang, Lihua; Peti-Peterdi, János; Bell, P Darwin; Schwiebert, Erik M

    2003-04-11

    Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

  16. Changes to channel sediments resulting from complex human impacts in a gravel-bed river, Polish Carpathians

    Science.gov (United States)

    Zawiejska, Joanna; Wyżga, Bartłomiej; Hajdukiewicz, Hanna; Radecki-Pawlik, Artur; Mikuś, Paweł

    2016-04-01

    deficit downstream as large volumes of finer bed material are flushed out from the incising channel section. Grain-size analyses of bulk gravels and measurements of 100 coarsest particles within the channel sediment ranging in age from 5200 years BP to the present, performed in this deeply incised section, indicated that grain size of channel sediments changed relatively little since mid-Holocene to the 1960s, but has increased rapidly over the last half-century as a result of human interventions and rapidly progressing channel incision. This study was performed within the scope of the Research Project DEC-2013/09/B/ST10/00056 financed by the National Science Centre of Poland.

  17. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    Science.gov (United States)

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  18. Molecular modeling and docking simulations of scorpion toxins and related analogs on human SKCa2 and SKCa3 channels.

    Science.gov (United States)

    Andreotti, Nicolas; di Luccio, Eric; Sampieri, François; De Waard, Michel; Sabatier, Jean-Marc

    2005-07-01

    The small-conductance Ca2+-activated K+ (SKCa) channels modulate cytosolic Ca2+ concentration in excitable and non-excitable tissues by regulating the membrane potential and are responsible of slow action potential after hyperpolarization that inhibits cell firing. Among these, human SKCa2 and SKCa3 channels differ in the pore region by only two residues: Ala331 and Asn367 (human small-conductance calcium-activated potassium channel, hSKCa2) instead of Val485 and His521 (hSKCa3). To design highly selective blockers of hSKCa channels, a number of known hSKCa2 and/or hSKCa3-active peptides (i.e. scorpion toxins and analogs thereof) were analyzed for their interactions and selectivities toward these channels. Molecular models of hSKCa2 and hSKCa3 channels (S5-H5-S6 portion) were generated, and scorpion toxins/peptides of unsolved three-dimensional (3D) structures were modeled. Models of toxin-channel complexes were generated by the bimolecular complex generation with global evaluation, and ranking (BiGGER) docking software and selected by using a screening method of the docking solutions. A high degree of correlation was found to exist between docking energies and experimental Kd values of peptides that blocked hSKCa2 and/or hSKCa3 channels, suggesting it could be appropriate to predict Kd values of other bioactive peptides. The best scoring complexes were also used to identify key residues of both interacting partners, indicating that such an approach should help the design of more active and/or selective peptide blockers of targeted ion channels.

  19. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells

    DEFF Research Database (Denmark)

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex

    2012-01-01

    as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors......The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined...... shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA...

  20. Origin and fate of the nucleolar channel system of normal human endometium

    Institute of Scientific and Technical Information of China (English)

    WANGTZUNENG; JSCHNEIDER

    1992-01-01

    Human normal endometrium was examined in ultrathin sections.Nucleolar channel system(NCS) appeared in the endometrial epithelial cells during the early and mid secretory phase of menstrual cycle.The NCS was a hollow ball like structure of different sizes and was composed of 2 to 5 rows of tubules embedded in an amporphous matrix.On its surface there were numerous electron dense particles resembling ribosomes,It was usually located within or associated with the nucleolus,SOmetimes,it was close to the nuclear envelope or protruding out from the nucleus .On occasion,NCS with simplified structure was found in the perinuclear cytoplasm.Concepts concerning the genesis,involution and function(s) of the NCS were disussed.

  1. Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans.

    Science.gov (United States)

    Wong, Brett J; Fieger, Sarah M

    2010-11-01

    The initial, rapid increase in skin blood flow in response to direct application of heat is thought to be mediated by an axon reflex, which is dependent on intact cutaneous sensory nerves. We tested the hypothesis that inhibition of transient receptor potential vanilloid type 1 (TRPV-1) channels, which are putative channels located on sensory nerves, would attenuate the skin blood flow response to local heating in humans. Ten subjects were equipped with four microdialysis fibres which were randomly assigned one of four treatments: (1) vehicle control (90% propylene glycol + 10% lactated Ringer solution); (2) 20 mm capsazepine to inhibit TRPV-1 channels; (3) 10 mm l-NAME to inhibit NO synthase; and (4) combined 20 mm capsazepine + 10 mm l-NAME. Following baseline measurements, the temperature of skin heaters was increased from 33°C to 42°C at a rate of 1.0°C every 10 s and local temperature was held at 42°C for 20-30 min until a stable plateau in skin blood flow was achieved. An index of skin blood flow was measured directly over each microdialysis site via laser-Doppler flowmetry (LDF). Beat-by-beat blood pressure was measured via photoplethysmography and verified via automated brachial auscultation. At the end of the local heating protocol, temperature of the heaters was increased to 43°C and 28 mm nitroprusside was infused to achieve maximal vasodilatation. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal values (%CVCmax). Initial peak in capsazepine (44 ± 4%CVCmax), l-NAME (56 ± 4%CVCmax) and capsazepine + l-NAME (32 ± 6%CVCmax) sites was significantly attenuated compared to control (87 ± 5%CVCmax; P thermal hyperaemia was significantly attenuated in capsazepine (73 ± 6%CVCmax), l-NAME (47 ± 5%CVCmax) and capsazepine + l-NAME (31 ± 7%CVCmax) sites compared to control (92 ± 5%CVCmax; P thermal hyperaemia. These data further suggest a portion of the NO component of thermal hyperaemia may be

  2. Fire history on the California Channel Islands spanning human arrival in the Americas.

    Science.gov (United States)

    Hardiman, Mark; Scott, Andrew C; Pinter, Nicholas; Anderson, R Scott; Ejarque, Ana; Carter-Champion, Alice; Staff, Richard A

    2016-06-05

    Recent studies have suggested that the first arrival of humans in the Americas during the end of the last Ice Age is associated with marked anthropogenic influences on landscape; in particular, with the use of fire which, would have given even small populations the ability to have broad impacts on the landscape. Understanding the impact of these early people is complicated by the dramatic changes in climate occurring with the shift from glacial to interglacial conditions. Despite these difficulties, we here attempt to test the extent of anthropogenic influence using the California Channel Islands as a smaller, landscape-scale test bed. These islands are famous for the discovery of the 'Arlington Springs Man', which are some of the earliest human remains in the Americas. A unifying sedimentary charcoal record is presented from Arlington Canyon, Santa Rosa Island, based on over 20 detailed sedimentary sections from eight key localities. Radiocarbon dating was based on thin, fragile, long fragments of charcoal in order to avoid the 'inbuilt' age problem. Radiocarbon dating of 49 such fragments has allowed inferences regarding the fire and landscape history of the Canyon ca 19-11 ka BP. A significant period of charcoal deposition is identified approximately 14-12.5 ka BP and bears remarkable closeness to an estimated age range of the first human arrival on the islands.This article is part of the themed issue 'The interaction of fire and mankind'.

  3. TRPM7 channel inhibition mediates midazolam-induced proliferation loss in human malignant glioma.

    Science.gov (United States)

    Chen, Jingkao; Dou, Yunling; Zheng, Xiaoke; Leng, Tiandong; Lu, Xiaofang; Ouyang, Ying; Sun, Huawei; Xing, Fan; Mai, Jialuo; Gu, Jiayu; Lu, Bingzheng; Yan, Guangmei; Lin, Jun; Zhu, Wenbo

    2016-11-01

    The melastatin-like transient receptor potential 7 (TRPM7) has been implicated in proliferation or apoptosis of some cancers, indicating the potential of TRPM7 as an anti-anaplastic target. Here, we identified the characteristic TRPM7 channel currents in human malignant glioma MGR2 cells, which could be blocked by a pharmacologic inhibitor Gd(3+). We mined the clinical sample data from Oncomine Database and found that human malignant glioma tissues expressed higher TRPM7 mRNA than normal brain ones. Importantly, we identified a widely used clinical anesthetic midazolam as a TRPM7 inhibitor. Midazolam treatment for seconds suppressed the TRPM7 currents and calcium influx, and treatment for 48 h inhibited the TRPM7 expression. The inhibitory effect on TRPM7 accounts for the proliferation loss and G0/G1 phase cell cycle arrest induced by midazolam. Our data demonstrates that midazolam represses proliferation of human malignant glioma cells through inhibiting TRPM7 currents, which may be further potentiated by suppressing the expression of TRPM7. Our result indicates midazolam as a pharmacologic lead compound with brain-blood barrier permeability for targeting TRPM7 in the glioma.

  4. Characterization and variation of a human inwardly-rectifying-K-channel gene (KCNJ6): a putative ATP-sensitive K-channel subunit.

    Science.gov (United States)

    Sakura, H; Bond, C; Warren-Perry, M; Horsley, S; Kearney, L; Tucker, S; Adelman, J; Turner, R; Ashcroft, F M

    1995-06-26

    The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta-cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP-sensitive K-channel expressed in brain and beta-cells, and characterisation of its exon-intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1-22.2). Analysis of single-stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro-149: CCG-CCA and Asp-328: GAC-GAT) with similar frequencies in normal and non-insulin-dependent diabetic patients.

  5. Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

    Science.gov (United States)

    Tarradas, Anna; Pinsach-Abuin, Mel Lina; Mackintosh, Carlos; Llorà-Batlle, Oriol; Pérez-Serra, Alexandra; Batlle, Montserrat; Pérez-Villa, Félix; Zimmer, Thomas; Garcia-Bassets, Ivan; Brugada, Ramon; Beltran-Alvarez, Pedro; Pagans, Sara

    2017-01-01

    Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel.

    Science.gov (United States)

    Liu, Zhenzhen; Jiang, Tianyu; Wang, Beilei; Ke, Bowen; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-02-02

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity.

  7. Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation

    Directory of Open Access Journals (Sweden)

    Anett Illing

    2013-01-01

    Full Text Available Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs generated from plucked human hair keratinocytes have been exploited in vitro to recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.

  8. Patch clamp studies of human sperm under physiological ionic conditions reveal three functionally and pharmacologically distinct cation channels.

    Science.gov (United States)

    Mansell, S A; Publicover, S J; Barratt, C L R; Wilson, S M

    2014-05-01

    Whilst fertilizing capacity depends upon a K(+) conductance (GK) that allows the spermatozoon membrane potential (Vm) to be held at a negative value, the characteristics of this conductance in human sperm are virtually unknown. We therefore studied the biophysical/pharmacological properties of the K(+) conductance in spermatozoa from normal donors held under voltage/current clamp in the whole cell recording configuration. Our standard recording conditions were designed to maintain quasi-physiological, Na(+), K(+) and Cl(-) gradients. Experiments that explored the effects of ionic substitution/ion channel blockers upon membrane current/potential showed that resting Vm was dependent upon a hyperpolarizing K(+) current that flowed via channels that displayed only weak voltage dependence and limited (∼7-fold) K(+) versus Na(+) selectivity. This conductance was blocked by quinidine (0.3 mM), bupivacaine (3 mM) and clofilium (50 µM), NNC55-0396 (2 µM) and mibefradil (30 µM), but not by 4-aminopyridine (2 mM, 4-AP). Progesterone had no effect upon the hyperpolarizing K(+) current. Repolarization after a test depolarization consistently evoked a transient inward 'tail current' (ITail) that flowed via a second population of ion channels with poor (∼3-fold) K(+) versus Na(+) selectivity. The activity of these channels was increased by quinidine, 4-AP and progesterone. Vm in human sperm is therefore dependent upon a hyperpolarizing K(+) current that flows via channels that most closely resemble those encoded by Slo3. Although 0.5 µM progesterone had no effect upon these channels, this hormone did activate the pharmacologically distinct channels that mediate ITail. In conclusion, this study reveals three functionally and pharmacologically distinct cation channels: Ik, ITail, ICatSper.

  9. A critical role for the transient receptor potential channel type 6 in human platelet activation.

    Directory of Open Access Journals (Sweden)

    Hari Priya Vemana

    Full Text Available While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6 mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules, integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.

  10. Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

    Science.gov (United States)

    Ase, Ariel R; Honson, Nicolette S; Zaghdane, Helmi; Pfeifer, Tom A; Séguéla, Philippe

    2015-04-01

    P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.

  11. Multi-channel microstrip transceiver arrays using harmonics for high field MR imaging in humans.

    Science.gov (United States)

    Wu, Bing; Wang, Chunsheng; Lu, Jonathan; Pang, Yong; Nelson, Sarah J; Vigneron, Daniel B; Zhang, Xiaoliang

    2012-02-01

    Radio-frequency (RF) transceiver array design using primary and higher order harmonics for in vivo parallel magnetic resonance imaging imaging (MRI) and spectroscopic imaging is proposed. The improved electromagnetic decoupling performance, unique magnetic field distributions and high-frequency operation capabilities of higher-order harmonics of resonators would benefit transceiver arrays for parallel MRI, especially for ultrahigh field parallel MRI. To demonstrate this technique, microstrip transceiver arrays using first and second harmonic resonators were developed for human head parallel imaging at 7T. Phantom and human head images were acquired and evaluated using the GRAPPA reconstruction algorithm. The higher-order harmonic transceiver array design technique was also assessed numerically using FDTD simulation. Compared with regular primary-resonance transceiver designs, the proposed higher-order harmonic technique provided an improved g-factor and increased decoupling among resonant elements without using dedicated decoupling circuits, which would potentially lead to a better parallel imaging performance and ultimately faster and higher quality imaging. The proposed technique is particularly suitable for densely spaced transceiver array design where the increased mutual inductance among the elements becomes problematic. In addition, it also provides a simple approach to readily upgrade the channels of a conventional primary resonator microstrip array to a larger number for faster imaging.

  12. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Waxman, S.G.

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pa

  13. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Waxman, S.G.

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pa

  14. Relaxant effect of a novel calcium-activated potassium channel modulator on human myometrial spontaneous contractility in vitro

    DEFF Research Database (Denmark)

    Rosenbaum, S.T.; Larsen, T.; Joergensen, J.C.

    2012-01-01

    Aim: To investigate the effect of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a novel SK/IK channels positive modulator, on human myometrial activity. Methods: Organ bath studies were performed on myometrial preparations obtained from women undergoing elective caesarean...

  15. The voltage-dependent K+ channels Kv1.3 and Kv1.5 in human cancer

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C.; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer. PMID:24133455

  16. The voltage-dependent K(+) channels Kv1.3 and Kv1.5 in human cancer.

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón Y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C; Felipe, Antonio

    2013-10-10

    Voltage-dependent K(+) channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer.

  17. OCT Study of Mechanical Properties Associated with Trabecular Meshwork and Collector Channel Motion in Human Eyes

    Science.gov (United States)

    Xin, Chen; Johnstone, Murray; Wang, Ningli; Wang, Ruikang K.

    2016-01-01

    We report the use of a high-resolution optical coherence tomography (OCT) imaging platform to identify and quantify pressure-dependent aqueous outflow system (AOS) tissue relationships and to infer mechanical stiffness through examination of tissue properties in ex vivo human eyes. Five enucleated human eyes are included in this study, with each eye prepared with four equal-sized quadrants, each encompassing 90 degrees of the limbal circumference. In radial limbal segments perfusion pressure within Schlemm’s canal (SC) is controlled by means of a perfusion cannula inserted into the canal lumen, while the other end of the cannula leads to a reservoir at a height that can control the pressure in the cannula. The OCT system images the sample with a spatial resolution of about 5 μm from the trabecular meshwork (TM) surface. Geometric parameters are quantified from the 2D OCT images acquired from the sample subjected to controlled changes in perfusion pressures; parameters include area and height of the lumen of SC, collector channel entrances (CCE) and intrascleral collector channels (ISCC). We show that 3D OCT imaging permits the identification of 3-D relationships of the SC, CCE and ISCC lumen dimensions. Collagen flaps or leaflets are found at CCE that are attached or hinged at only one end, whilst the flaps are connected to the TM by cylindrical structures spanning SC. Increasing static SC pressures resulted in SC lumen enlargement with corresponding enlargement of the CCE and ISCC lumen. Pressure-dependent SC lumen area and height changes are significant at the 0.01 levels for ANOVA, and at the 0.05 for both polynomial curves and Tukey paired comparisons. Dynamic measurements demonstrate a synchronous increase in SC, CCE and ISCC lumen height in response to pressure changes from 0 to 10, 30 or 50 mm Hg, respectively, and the response time is within the 50-millisecond range. From the measured SC volume and corresponding IOP values, we demonstrate that an

  18. NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.

    Science.gov (United States)

    Paramonov, A S; Lyukmanova, E N; Myshkin, M Yu; Shulepko, M A; Kulbatskii, D S; Petrosian, N S; Chugunov, A O; Dolgikh, D A; Kirpichnikov, M P; Arseniev, A S; Shenkarev, Z O

    2017-03-01

    Voltage-gated Na(+) channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na(+) channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na(+) channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of (13)C,(15)N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na(+) channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn(2+) titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. (15)N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K(+) channels. These results validate structural studies of isolated VSDs of Na(+) channels and show possible pitfalls in application of this 'divide and conquer' approach.

  19. Activator-induced dynamic disorder and molecular memory in human two-pore domain hTREK1 K channel.

    Science.gov (United States)

    Nayak, Tapan Kumar; Dana, Saswati; Raha, Soumyendu; Sikdar, Sujit K

    2011-04-01

    Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. Ion channel flux activity is comparable to the catalytic activity of enzyme molecules. Saturating concentrations of substrate induce "dynamic disorder" in the kinetic rate processes of single-enzyme molecules and consequently, develop correlative "memory" of the previous history of activities. Similarly, binding of ions as substrate alone or in presence of agonists affects the catalytic turnover of single-ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in the single human-TREK1-channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that the single-hTREK1-channel behaves as a typical Michaelis-Menten enzyme molecule with a high-affinity binding site for K(+) ion as substrate. But, in contrast to enzyme, dynamic disorder in single-hTREK1-channel was not induced by substrate K(+) binding, but required allosteric modification of the channel molecule by the agonist, trichloroethanol. In addition, interaction of trichloroethanol with hTREK1 induced strong correlation in the waiting time and flux intensity, exemplified by distinct mode-switching between high and low flux activities. This suggested the induction of molecular memory in the channel molecule by the agonist, which persisted for several decades in time. Our mathematical modeling studies identified the kinetic rate processes associated with dynamic disorder. It further revealed the presence of multiple populations of distinct conformations that contributed to the "heterogeneity" and consequently, to the molecular memory phenomenon that we observed. The online version of this article (doi:10.1007/s12154-010-0053-3) contains supplementary material, which is available to authorized users.

  20. High affinity complexes of pannexin channels and L-type calcium channel splice-variants in human lung: Possible role in clevidipine-induced dyspnea relief in acute heart failure

    Directory of Open Access Journals (Sweden)

    Gerhard P. Dahl

    2016-08-01

    Research in Context: Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure.

  1. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    Science.gov (United States)

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  2. Characterisation of K+ Channels in Human Fetoplacental Vascular Smooth Muscle Cells

    OpenAIRE

    Brereton, Melissa F.; Mark Wareing; Rebecca L Jones; Greenwood, Susan L.

    2013-01-01

    Adequate blood flow through placental chorionic plate resistance arteries (CPAs) is necessary for oxygen and nutrient transfer to the fetus and a successful pregnancy. In non-placental vascular smooth muscle cells (SMCs), K(+) channels regulate contraction, vascular tone and blood flow. Previous studies showed that K(+) channel modulators alter CPA tone, but did not distinguish between effects on K(+) channels in endothelial cells and SMCs. In this study, we developed a preparation of freshly...

  3. Arecoline inhibits intermediate-conductance calcium-activated potassium channels in human glioblastoma cell lines.

    Science.gov (United States)

    So, Edmund Cheung; Huang, Yan-Ming; Hsing, Chung-Hsi; Liao, Yu-Kai; Wu, Sheng-Nan

    2015-07-05

    Arecoline (ARE) is an alkaloid-type natural product from areca nut. This compound has numerous pharmacological and toxicological effects. Whether this agent interacts with ion channels to perturb functional activity of cells remains unknown. The effects of ARE on ionic currents were studied in glioma cell lines (U373 and U87MG) using patch-clamp technique. Like TRAM-34(1-[(2-chlorophenyl)-diphenylmethyl]pyrazole), ARE suppressed the amplitude of whole-cell voltage-gated K(+) currents in U373 cells elicited by a ramp voltage clamp. In cell-attached configuration, ARE did not modify the single-channel conductance of intermediate-conductance Ca(2+)-activated K(+) (IKCa) channels; however, it did reduce channel activity. Its inhibition of IKCa channels was accompanied by a significant lengthening in the slow component of mean closed time of IKCa channels. Based on minimal kinetic scheme, the dissociation constant (KD) required for ARE-mediated prolongation of mean closed time was 11.2µM. ARE-induced inhibition of IKCa channels was voltage-dependent. Inability of ARE to perturb the activity of large-conductance Ca(2+)-activated K(+) (BKCa) channels was seen. Under current-clamp recordings, ARE depolarized the membrane of U373 cells and DCEBIO reversed ARE-induced depolarization. Similarly, ARE suppressed IKCa-channel activities in oral keratinocytes. This study provides the evidence that ARE block IKCa channels in a concentration, voltage and state-dependent manner. ARE-induced block of IKCa channels is unrelated to the binding of muscarinic receptors. The effects of ARE on these channels may partially be responsible for the underlying cellular mechanisms by which it influences the functional activities of glioma cells or oral keratinocytes, if similar findings occur in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    Science.gov (United States)

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  5. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    Science.gov (United States)

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  6. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    Science.gov (United States)

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  7. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    Directory of Open Access Journals (Sweden)

    Cui Zhu

    2016-08-01

    Full Text Available Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11 have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes, goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  8. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    Directory of Open Access Journals (Sweden)

    Niv Bachnoff

    2011-01-01

    Full Text Available A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s at the C-tail of α11.2, the pore forming subunit of CaV1.2.

  9. Effect of dexamethasone on voltage-gated Na+ channel in cultured human bronchial smooth muscle cells.

    Science.gov (United States)

    Nakajima, Toshiaki; Jo, Taisuke; Meguro, Kentaro; Oonuma, Hitoshi; Ma, Ji; Kubota, Nami; Imuta, Hiroyuki; Takano, Haruhito; Iida, Haruko; Nagase, Takahide; Nagata, Taiji

    2008-06-06

    Voltage-gated Na(+) channel (I(Na)) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I(Na), by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10(-6) M) did not affect I(Na). However, the percentage of the cells with I(Na) was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I(Na) adjusted by cell capacitance in cells with I(Na) was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that alpha and beta subunits mRNA of I(Na) mainly consisted of SCN9A and SCN1beta, respectively. Treatment with dexamethasone for 24-48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I(Na) encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor.

  10. Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

    DEFF Research Database (Denmark)

    Kroigaard, Christel; Dalsgaard, Thomas; Nielsen, Gorm

    2012-01-01

    BACKGROUND AND PURPOSE: Small (K(Ca) 2) and intermediate (K(Ca) 3.1) conductance calcium-activated potassium channels (K(Ca) ) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we...... investigated the expression of K(Ca) 2.3 and K(Ca) 3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. EXPERIMENTAL APPROACH: Channel expression and functional studies were conducted in human isolated small pulmonary arteries.......1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K(Ca) 2 channel blocker apamin, while the K(Ca) 3.1 channel blocker, charybdotoxin failed to reduce...

  11. [Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

    Science.gov (United States)

    Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

    2012-01-01

    Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

  12. I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

    Science.gov (United States)

    Scheruebel, Susanne; Koyani, Chintan N; Hallström, Seth; Lang, Petra; Platzer, Dieter; Mächler, Heinrich; Lohner, Karl; Malle, Ernst; Zorn-Pauly, Klaus; Pelzmann, Brigitte

    2014-07-01

    Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

  13. Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na(+) channel inactivation.

    Science.gov (United States)

    Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

    2017-01-01

    In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na(+) currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  14. First evidence of TRPV5 and TRPV6 channels in human parathyroid glands: possible involvement in neoplastic transformation.

    Science.gov (United States)

    Giusti, Laura; Cetani, Filomena; Da Valle, Ylenia; Pardi, Elena; Ciregia, Federica; Donadio, Elena; Gargini, Claudia; Piano, Ilaria; Borsari, Simona; Jaber, Ali; Caputo, Antonella; Basolo, Fulvio; Giannaccini, Gino; Marcocci, Claudio; Lucacchini, Antonio

    2014-10-01

    The parathyroid glands play an overall regulatory role in the systemic calcium (Ca(2+)) homeostasis. The purpose of the present study was to demonstrate the presence of the Ca(2+) channels transient receptor potential vanilloid (TRPV) 5 and TRPV6 in human parathyroid glands. Semi-quantitative and quantitative PCR was carried out to evaluate the presence of TRPV5 and TRPV6 mRNAs in sporadic parathyroid adenomas and normal parathyroid glands. Western blot and immunocytochemical assays were used to assess protein expression, cellular localization and time expression in primary cultures from human parathyroid adenoma. TRPV5 and TRPV6 transcripts were then identified both in normal and pathological tissues. Predominant immunoreactive bands were detected at 75-80 kD for both vanilloid channels. These channels co-localized with the calcium-sensing receptor (CASR) on the membrane surface, but immunoreactivity was also detected in the cytosol and around the nuclei. Our data showed that western blotting recorded an increase of protein expression of both channels in adenoma samples compared with normal glands suggesting a potential relation with the cell calcium signalling pathway and the pathological processes of these glands.

  15. The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida

    Institute of Scientific and Technical Information of China (English)

    Julio C Chávez; Claudia L Trevi(n)o; Gerardo A de Blas; José L de la Vega-Beltrán; Takuya Nishigaki; Mayel Chirinos; María Elena González-González; Fernando Larrea; Alejandra Solís; Alberto Darszon

    2011-01-01

    The acrosome reaction(AR),an absolute requirement for spermatozoa and egg fusion,requires the influx of Ca2+into the spermatozoa through voltage-dependent Ca2+channels and store-operated channels.Maitotoxin(MTx),a Ca2+-mobilizing agent,has been shown to be a potent inducer of the mouse sperm AR,with a pharmacology similar to that of the zona pellucida(ZP),possibly suggesting a common pathway for both inducers.Using recombinant human ZP3(rhZP3),mouse ZP and two MTx channel blockers(U73122 and U73343),we investigated and compared the MTx-and ZP-induced ARs in human and mouse spermatozoa.Herein,we report that MTx induced AR and elevated intracellular Ca2+([Ca2+]1)in human spermatozoa,both of which were blocked by U73122 and U73343.These two compounds also inhibited the MTx-induced AR in mouse spermatozoa.In disagreement with our previous proposal,the AR triggered by rhZP3 or mouse ZP was not blocked by U73343,indicating that in human and mouse spermatozoa,the AR induction by the physiologicalligands or by MTx occurred through distinct pathways.U73122,but not U73343(inactive analogue),can block phospholipase C(PLC).Another PLC inhibitor,edelfosine,also blocked the rhZP3-and ZP-induced ARs.These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR.Notably,edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human,suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

  16. A user-friendly wearable single-channel EOG-based human-computer interface for cursor control

    OpenAIRE

    2015-01-01

    This paper presents a novel wearable single-channel electrooculography (EOG) based human-computer interface (HCI) with a simple system design and robust performance. In the proposed system, EOG signals for control are generated from double eye blinks, collected by a commercial wearable device (the NeuroSky MindWave headset), and then converted into a sequence of commands that can control cursor navigations and actions. The EOG-based cursor control system was tested on 8 subjects in indoor or ...

  17. How Human Resource Professionals Use Electronic Channels to Communicate CSR : A case study focused on Solvay's French industrial sites

    OpenAIRE

    Fournet, Clara; Pauly, Marissa

    2015-01-01

    Corporate Social Responsibility (CSR) has become a large concern for many companies with the rise of globalization. Oftentimes, companies are encouraged to communicate CSR externally, but not internally. This research focuses upon the internal communication of CSR, specifically how Human Resource (HR) professionals use electronic channels to communicate to employees. The scope of this research is focused solely upon HR professionals within Solvay’s French industrial sites, which produce chemi...

  18. Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

    Energy Technology Data Exchange (ETDEWEB)

    Inada, Hitoshi; Procko, Erik; Sotomayor, Marcos; Gaudet, Rachelle (Harvard-Med); (Harvard)

    2012-10-23

    The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

  19. Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

    Directory of Open Access Journals (Sweden)

    Reesha R Patel

    Full Text Available Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs. Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have

  20. The Realization of Command Channel-Mechanism in a System for Human to Human Interaction and Co-operative%人人交互与协作系统中命令通道机制的实现

    Institute of Scientific and Technical Information of China (English)

    田友胜; 侯义斌

    2000-01-01

    The concept of command channel-mechanism has been introduced into the command channelmechanism system for human to human interaction and co-operative over the Internet/Intranet.A new type of interaction and co-operative environment has been built up.This paper discusses the running mechanism and software realization of the command channel.

  1. Modulation of the transient outward current (Ito) in rat cardiac myocytes and human Kv4.3 channels by mefloquine.

    Science.gov (United States)

    Perez-Cortes, E J; Islas, A A; Arevalo, J P; Mancilla, C; Monjaraz, E; Salinas-Stefanon, E M

    2015-10-15

    The antimalarial drug mefloquine, is known to be a potassium channel blocker, although its mechanism of action has not being elucidated and its effects on the transient outward current (Ito) and the molecular correlate, the Kv4.3 channel has not being studied. Here, we describe the mefloquine-induced inhibition of the rat ventricular Ito and of CHO cells co-transfected with human Kv4.3 and its accessory subunit hKChIP2C by whole-cell voltage-clamp. Mefloquine inhibited rat Ito and hKv4.3+KChIP2C currents in a concentration-dependent manner with a limited voltage dependence and similar potencies (IC50=8.9μM and 10.5μM for cardiac myocytes and Kv4.3 channels, respectively). In addition, mefloquine did not affect the activation of either current but significantly modified the hKv4.3 steady-state inactivation and recovery from inactivation. The effects of this drug was compared with that of 4-aminopyridine (4-AP), a well-known potassium channel blocker and its binding site does not seem to overlap with that of 4-AP.

  2. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from t

  3. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway

    DEFF Research Database (Denmark)

    Liu, Daoyan; Maier, Alexandra; Scholze, Alexandra;

    2008-01-01

    Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels....

  4. Block of human cardiac sodium channels by lacosamide: evidence for slow drug binding along the activation pathway.

    Science.gov (United States)

    Wang, Ging Kuo; Wang, Sho-Ya

    2014-05-01

    Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na(+) channels for its therapeutic action. Cardiac Na(+) channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na(+) channels. Lacosamide showed little effect on hNav1.5 Na(+) currents at 300 µM when cells were held at -140 mV. With 30-second conditioning pulses from -90 to -50 mV; however, hNav1.5 Na(+) channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70-80 µM. Higher IC50 values were found at -110 and -30 mV. The development of lacosamide block at -70 mV was slow in wild-type Na(+) channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at -70 mV for 10 seconds was relatively rapid in wild-type Na(+) channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na(+) currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na(+) channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na(+) channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway.

  5. Nonlocal atlas-guided multi-channel forest learning for human brain labeling

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Guangkai [Space Control and Inertial Technology Research Center, Harbin Institute of Technology, Harbin 150001, China and Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 (United States); Gao, Yaozong; Wu, Guorong [Department of Computer Science, Department of Radiology, and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 (United States); Wu, Ligang [Space Control and Inertial Technology Research Center, Harbin Institute of Technology, Harbin 150001 (China); Shen, Dinggang, E-mail: dgshen@med.unc.edu [Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and Department of Brain and Cognitive Engineering, Korea University, Seoul 02841 (Korea, Republic of)

    2016-02-15

    Purpose: It is important for many quantitative brain studies to label meaningful anatomical regions in MR brain images. However, due to high complexity of brain structures and ambiguous boundaries between different anatomical regions, the anatomical labeling of MR brain images is still quite a challenging task. In many existing label fusion methods, appearance information is widely used. However, since local anatomy in the human brain is often complex, the appearance information alone is limited in characterizing each image point, especially for identifying the same anatomical structure across different subjects. Recent progress in computer vision suggests that the context features can be very useful in identifying an object from a complex scene. In light of this, the authors propose a novel learning-based label fusion method by using both low-level appearance features (computed from the target image) and high-level context features (computed from warped atlases or tentative labeling maps of the target image). Methods: In particular, the authors employ a multi-channel random forest to learn the nonlinear relationship between these hybrid features and target labels (i.e., corresponding to certain anatomical structures). Specifically, at each of the iterations, the random forest will output tentative labeling maps of the target image, from which the authors compute spatial label context features and then use in combination with original appearance features of the target image to refine the labeling. Moreover, to accommodate the high inter-subject variations, the authors further extend their learning-based label fusion to a multi-atlas scenario, i.e., they train a random forest for each atlas and then obtain the final labeling result according to the consensus of results from all atlases. Results: The authors have comprehensively evaluated their method on both public LONI-LBPA40 and IXI datasets. To quantitatively evaluate the labeling accuracy, the authors use the

  6. Search after new agents for hyperpolarization-activated and cyclic nucleotide-gated ion channels; Suche nach neuen Wirkstoffen fuer Hyperpolarisationsaktivierte und zyklisch Nukleotid-gesteuerte Ionenkanaele

    Energy Technology Data Exchange (ETDEWEB)

    Struenker, T.

    2005-12-01

    Rhythmic activity of single cells or cellular networks is a common feature of most organisms. Cellular rhythms govern the beating of the heart, cycles of sleep and wakefulness, breathing, and the release of hormones. The endogenous rhythmic activity of many neurons and cardiac relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity because it determines the frequency of the oscillations. The activity of the channels is thus setting the ''pace'' of the activity; therefore, these channels are often referred to as ''pacemaker'' channels. Despite their obvious physiological importance it hasn't been until a few years ago that the genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. From a scientific as well as medical point of view, HCN channels are interesting drug targets. Only a few substances are known that specifically affect HCN channels. In the present study, a microtiter plate-based high throughput screening assay for HCN1 and HCN4 channels was developed. With this assay, known drugs for HCN channels were characterized. Subsequently, venoms of snails, spiders, scorpions, and snakes were screened for toxins affecting HCN channel activity. A few venoms were identified that possibly contain drugs that act on HCN channels. (orig.)

  7. Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity.

    Science.gov (United States)

    Bury, M; Girault, A; Mégalizzi, V; Spiegl-Kreinecker, S; Mathieu, V; Berger, W; Evidente, A; Kornienko, A; Gailly, P; Vandier, C; Kiss, R

    2013-03-28

    Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca(2+)-activated K(+) channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.

  8. The Piezo1 protein ion channel functions in human nucleus pulposus cell apoptosis by regulating mitochondrial dysfunction and the endoplasmic reticulum stress signal pathway.

    Science.gov (United States)

    Li, Xiao-Fei; Leng, Ping; Zhang, Zhao; Zhang, Hai-Ning

    2017-09-15

    The Piezo1 protein ion channel is a novel mechanical stretch-activated ion channel (SAC) closely related to mechanical signals. Mechanotransduction plays a crucial role in organ development and homeostasis. Previous studies identified Piezo1 and demonstrated that it is distinct from other ion channels with well-established roles in lower organisms. Mechanical stretch-activated ion channels from other organisms are not conserved in mammals or do not act as mechanically activated channels in mammals. In the current study, we explored the role of the Piezo1 ion channel in human nucleus pulposus cell (NP cell) apoptosis through mechanical force-induced mitochondrial dysfunction and endoplasmic reticulum stress. Reverse Transcription Polymerase chain reaction (RT-PCR), immunofluorescence, immunohistochemistry and Annexin V binding and propidium iodide analyses revealed that the Piezo1 protein ion channel was highly expressed in human NP cells, which are the primary cells that comprise the intervertebral disc. In patients with intervertebral disc degeneration (IVDD), the Piezo1 protein may play a crucial role in human NP cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress under abnormal loading conditions. This study also verified that human NP cells have an intimate connection with the cytoskeleton upon treatment of the cells with the Piezo1 blocking peptide GsMTx4 from tarantula venom. In summary, Piezo1 functions in human NP cell apoptosis, which may be one underlying mechanism of apoptosis induced by abnormal loading in IVDD patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Natural and human-induced driving factors in the evolution of tidal channels: case studies in the Venice Lagoon (Italy).

    Science.gov (United States)

    Rizzetto, Federica

    2013-04-01

    Coastal wetlands are largely affected by a complex variety of both natural and anthropogenic factors, which induce evident, often irreversible, geomorphological transformations. In particular, this research focuses on the main processes that influence the evolution of tidal channels in salt marshes and shows the results derived from the analysis of some case studies in the Venice Lagoon (northwestern Adriatic Sea, Italy). Here tidal network has been recognized as significantly sensitive to sea-level rise and tide oscillations (Rizzetto and Tosi, 2011; Rizzetto and Tosi, 2012), but it is also vulnerable to human impact. The sites were selected in areas characterized by low anthropogenic pressure to prevent strong human interferences from completely masking the effects of natural forces. The interpretation of a large number of high-resolution aerial photographs, taken since the mid 1930s, allowed identifying in detail tidal channel evolution, both in the long- and in the short-term. The observation of historical and recent topographic maps completed the study and provided other important data to define the modifications occurred in the past two centuries. The channel planform changes were determined through the morphometric analysis of the tidal network, carried out using a Geographic Information System software. These modifications were interpreted in the light of sea-level oscillations (i.e. relative sea-level rise and strength/frequency of high tides, which are increasing owing to climate changes), variations of sediment supply, and human activities occurred in the past century. The joint analysis of all the data allowed distinguishing the changes induced by both relative sea-level rise and high tides on planform pattern and evolution of tidal channels, and identifying the effects of human interferences, which magnified the impact of natural factors (e.g. groundwater exploitation responsible for high subsidence rates between 1950 and 1970 and, consequently, for an

  10. Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells.

    LENUS (Irish Health Repository)

    Muchekehu, Ruth W

    2009-02-01

    Steroid hormones target K+ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

  11. Effects on Humans Elicited by Inhaling the Fragrance of Essential Oils: Sensory Test, Multi-Channel Thermometric Study and Forehead Surface Potential Wave Measurement on Basil and Peppermint

    National Research Council Canada - National Science Library

    SATOH, Tomoko; SUGAWARA, Yoshiaki

    2003-01-01

    The effects on humans inhaling the fragrance of essential oils were examined in terms of a sensory test, a multi-channel skin thermometer study and a portable forehead surface electroencephalographic (IBVA-EEG) measurement...

  12. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    Science.gov (United States)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  13. Effect of beta-adrenoceptor blockers on human ether-a-go-go-related gene (HERG) potassium channels

    DEFF Research Database (Denmark)

    Dupuis, Delphine S; Klaerke, Dan A; Olesen, Søren-Peter

    2005-01-01

    Patients with congenital long QT syndrome may develop arrhythmias under conditions of increased sympathetic tone. We have addressed whether some of the beta-adrenoceptor blockers commonly used to prevent the development of these arrhythmias could per se block the cardiac HERG (Human Ether....... These data showed that HERG blockade by beta-adrenoceptor blockers occurred only at high micromolar concentrations, which are significantly above the recently established safe margin of 100 (Redfern et al., 2003).......-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) blocked the HERG channel with similar affinity, whereas the beta1-receptor antagonists metoprolol and atenolol showed weak effects. Further, the four compounds blocked HERG channels expressed in a mammalian HEK293 cell line...

  14. Computational analysis of the effects of the hERG channel opener NS1643 in a human ventricular cell model

    DEFF Research Database (Denmark)

    Peitersen, Torben; Grunnet, Morten; Benson, Alan P

    2008-01-01

    BACKGROUND: Dysfunction or pharmacologic inhibition of repolarizing cardiac ionic currents can lead to fatal arrhythmias. The hERG potassium channel underlies the repolarizing current I(Kr), and mutations therein can produce both long and short QT syndromes (LQT2 and SQT1). We previously reported...... on the diphenylurea compound NS1643, which acts on hERG channels in two distinct ways: by increasing overall conductance and by shifting the inactivation curve in the depolarized direction. OBJECTIVE: The purpose of this study was to determine which of the two components contributes more to the antiarrhythmic effects...... of NS1643 under normokalemic and hypokalemic conditions. METHODS: The study consisted of mathematical simulation of action potentials in a human ventricular ionic cell model in single cell and string of 100 cells. RESULTS: Regardless of external potassium concentration or diastolic interval used, NS1643...

  15. Voltage-gated sodium channel expressed in cultured human smooth muscle cells: involvement of SCN9A.

    Science.gov (United States)

    Jo, Taisuke; Nagata, Taiji; Iida, Haruko; Imuta, Hiroyuki; Iwasawa, Kuniaki; Ma, Ji; Hara, Kei; Omata, Masao; Nagai, Ryozo; Takizawa, Hajime; Nagase, Takahide; Nakajima, Toshiaki

    2004-06-04

    Voltage-gated Na(+) channel (I(Na)) is expressed under culture conditions in human smooth muscle cells (hSMCs) such as coronary myocytes. The aim of this study is to clarify the physiological, pharmacological and molecular characteristics of I(Na) expressed in cultured hSMCs obtained from bronchus, main pulmonary and coronary artery. I(Na), was recorded in these hSMCs and inhibited by tetrodotoxin (TTX) with an IC(50) value of approximately 10 nM. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of mRNA showed the prominent expression of transcripts for SCN9A, which was consistent with the results of real-time quantitative RT-PCR. These results provide novel evidence that TTX-sensitive Na(+) channel expressed in cultured hSMCs is mainly composed of Na(v)1.7.

  16. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2008-11-01

    The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry\\/signalling pathway that is sensitive to 17beta-estradiol.

  17. Single Particle Image Reconstruction of the Human Recombinant Kv2.1 Channel

    OpenAIRE

    Adair, Brian; Nunn, Rashmi; Lewis, Shannon; Dukes, Iain; Philipson, Louis; Yeager, Mark

    2008-01-01

    Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K+ currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic β-cells. They are therefore an important...

  18. Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women.

    Science.gov (United States)

    Reinl, Erin L; Cabeza, Rafael; Gregory, Ismail A; Cahill, Alison G; England, Sarah K

    2015-10-01

    Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.

  19. Reach-scale channel sensitivity to multiple human activities and natural events: Lower Santa Clara River, California, USA

    Science.gov (United States)

    Downs, Peter W.; Dusterhoff, Scott R.; Sears, William A.

    2013-05-01

    Understanding the cumulative impact of natural and human influences on the sensitivity of channel morphodynamics, a relative measure between the drivers for change and the magnitude of channel response, requires an approach that accommodates spatial and temporal variability in the suite of primary stressors. Multiple historical data sources were assembled to provide a reach-scale analysis of the lower Santa Clara River (LSCR) in Ventura County, California, USA. Sediment supply is naturally high due to tectonic activity, earthquake-generated landslides, wildfires, and high magnitude flow events during El Niño years. Somewhat typically for the region, the catchment has been subject to four reasonably distinct land use and resource management combinations since European-American settlement. When combined with analysis of channel morphological response (quantifiable since ca. 1930), reach-scale and temporal differences in channel sensitivity become apparent. Downstream reaches have incised on average 2.4 m and become narrower by almost 50% with changes focused in a period of highly sensitive response after about 1950 followed by forced insensitivity caused by structural flood embankments and a significant grade control structure. In contrast, the middle reaches have been responsive but are morphologically resilient, and the upstream reaches show a mildly sensitive aggradational trend. Superimposing the natural and human drivers for change reveals that large scale stressors (related to ranching and irrigation) have been replaced over time by a suite of stressors operating at multiple spatial scales. Lower reaches have been sensitive primarily to 'local' scale impacts (urban growth, flood control, and aggregate mining) whereas, upstream, catchment-scale influences still prevail (including flow regulation and climate-driven sediment supply factors). These factors illustrate the complexity inherent to cumulative impact assessment in fluvial systems, provide evidence for a

  20. Heterologous expression and purification of an active human TRPV3 ion channel

    DEFF Research Database (Denmark)

    Kol, Stefan; Braun, Christian; Thiel, Gerhard

    2013-01-01

    selected a suitable detergent and buffer system using analytical size‐exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α‐helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also...

  1. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  2. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  3. Human endogenous retrovirus W family envelope gene activates the small conductance Ca2+-activated K+ channel in human neuroblastoma cells through CREB.

    Science.gov (United States)

    Li, S; Liu, Z C; Yin, S J; Chen, Y T; Yu, H L; Zeng, J; Zhang, Q; Zhu, F

    2013-09-05

    Numerous studies have shown that human endogenous retrovirus W family (HERV-W) envelope gene (env) is related to various diseases but the underlying mechanism has remained poorly understood. Our previous study showed that there was abnormal expression of HERV-W env in sera of patients with schizophrenia. In this paper, we reported that overexpression of the HERV-W env elevated the levels of small conductance Ca(2+)-activated K(+) channel protein 3 (SK3) in human neuroblastoma cells. Using a luciferase reporter system and RNA interference method, we found that functional cAMP response element site was required for the expression of SK3 triggered by HERV-W env. In addition, it was also found that the SK3 channel was activated by HERV-W env. Further study indicated that cAMP response element-binding protein (CREB) was required for the activation of the SK3 channel. Thus, a novel signaling mechanism of how HERV-W env influences neuronal activity and contributes to mental illnesses such as schizophrenia was proposed.

  4. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Soo Hwa [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Choi, Changsun [Department of Food and Nutrition, College of Human Ecology, Chung-Ang University, Anseong, Gyeonggi (Korea, Republic of); Hong, Seong-Geun; Yarishkin, Oleg V. [Department of Physiology, College of Medicine, Gyeongsang National University, Jinju (Korea, Republic of); Bae, Young Min; Kim, Jae Gon [Department of Physiology, College of Medicine, Konkuk University, Seoul (Korea, Republic of); O' Grady, Scott M. [Department of Physiology, 495 Animal Science/Veterinary Medicine Bldg., St. Paul, University of Minnesota, MN (United States); Yoon, Kyong-Ah [Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi (Korea, Republic of); Kang, Kyung-Sun [Veterinary Public Health, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul (Korea, Republic of); Ryu, Pan Dong [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of)

    2009-06-26

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.

  5. Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia.

    Science.gov (United States)

    Hamann, Melanie; Meisler, Miriam H; Richter, Angelika

    2003-12-01

    The med(J) mouse with twisting movements related to deficiency of the sodium channel Scn8a has been proposed as a model of kinesiogenic dystonia. This prompted us to examine the phenotype of these mice in more detail. By cortical electroencephalographic (EEG) recordings, we could not detect any changes, demonstrating that the motor disturbances are not epileptic in nature, an important similarity to human dystonia. The significantly decreased body weight of med(J) mice was related to reduced food intake. Observations in the open field and by video recordings revealed that the mice exhibit sustained abnormal postures and movements of limbs, trunk and tail not only during locomotor activity but also at rest. With the exception of the head tremor, the other motor impairments were persistent rather than paroxysmal. When several neurological reflexes were tested, alterations were restricted to the posture and righting reflexes. Results of the wire hang test confirmed the greatly reduced muscle strength in the med(J) mouse. In agreement with different types of human dystonia, biperiden, haloperidol and diazepam moderately reduced the severity of motor disturbances in med(J) mice. In view of the sodium channel deficiency in med(J) mice, the beneficial effects of the sodium channel blocker phenytoin was an unexpected finding. By immunohistochemical examinations, the density of nigral dopaminergic neurons was found to be unaltered, substantiating the absence of pathomorphological abnormalities within the brain of med(J) mice shown by previous studies. With the exception of muscle weakness, many of the features of the med(J) mouse are similar to human idiopathic dystonia.

  6. The mystery is solved-CatSper is the principal calcium channel activated by progesterone in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Christopher LR Barratt

    2011-01-01

    @@ Aremarkable advance in sperm physiology has recently been published in Nature.Two groups using patch clamping techniques on human sperm have solved a mystery about the sperm cell that has puzzled both andrologists and those involved in non-genomic cellular signalling for over 20 years.In these papers, Lishko1 and Strunker2 independently demonstrate that the universal characteristic effect of progesterone on sperm-a rapid influx of calcium-is via a sperm-specific channel CatSper.

  7. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders

    OpenAIRE

    Drenth, J.P.H.; Waxman, S G

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Na(v)1.7 have been shown to cause primary erythermalgia and paroxysmal extreme pain disorder, while nonsense mutations in Na(v)1.7 result in los...

  8. FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells.

    Science.gov (United States)

    López, Esther; Berna-Erro, Alejandro; Salido, Ginés M; Rosado, Juan A; Redondo, Pedro C

    2013-03-01

    Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca(2+) channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca(2+) entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca(2+)](c)), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca(2+) mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn(2+) entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP(3)R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.

  9. Structural analysis of the S4-S5 linker of the human KCNQ1 potassium channel.

    Science.gov (United States)

    Gayen, Shovanlal; Li, Qingxin; Kang, CongBao

    2015-01-02

    KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4-S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4-S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4-S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane.

  10. Modeling hysteresis observed in the human erythrocyte voltage-dependent cation channel

    DEFF Research Database (Denmark)

    Flyvbjerg, Henrik; Gudowska-Nowak, Ewa; Christophersen, Palle

    2012-01-01

    cycle, including its direction, is reproduced by a model with 2×2 discrete states: the normal open/closed states and two different states of "gate tension". Rates of transitions between the two branches of the hysteresis curve are modeled with single-barrier kinetics by introducing a real......-valued "reaction coordinate" parametrizing the protein's conformational change between the two states of gate tension. The resulting scenario suggests a reanalysis of former experiments with NSVDC channels....

  11. Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny

    Directory of Open Access Journals (Sweden)

    Leonhard Linta

    2013-01-01

    Full Text Available Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

  12. Latest Holocene evolution and human disturbance of a channel segment in the Hudson River Estuary

    Science.gov (United States)

    Klingbeil, A.D.; Sommerfield, C.K.

    2005-01-01

    The latest Holocene sedimentary record of a cohesive channel and subtidal shoal in the lower Hudson River Estuary was examined to elucidate natural (sea-level rise, sediment transport) and anthropogenic (bulkheading, dredging) influences on the recent morphodynamic evolution of the system. To characterize the seafloor and shallow subbottom, ??? 100 km of high-resolution seismic reflection profiles (chirp) were collected within a 20-km reach of the estuary and correlated with sediment lithologies provided by eight vibracores recovered along seismic lines. Sediment geochronology with 137Cs and 14C was used to estimate intermediate and long-term sedimentation rates, respectively, and historical bathymetric data were analyzed to identify regional patterns of accretion and erosion, and to quantify changes in channel geometry and sediment volume. The shoal lithosome originated around 4 ka presumably with decelerating eustatic sea level rise during the latest Holocene. Long-term sedimentation rates on the shoal (2.3-2.6 mm/yr) are higher than in the channel (2 mm/yr) owing to hydrodynamic conditions that preferentially sequester suspended sediment on the western side of the estuary. As a result, the shoal accretes oblique to the principal axis of tidal transport, and more rapidly than the channel to produce an asymmetric cross-section. Shoal deposits consist of tidally bedded muds and are stratified by minor erosion surfaces that seismic profiles reveal to extend for 10s of meters to kilometers. The frequency and continuity of these surfaces suggest that the surficial shoal is catastrophically stripped on decadal-centennial time scales by elevated tidal flows; tidal erosion maintains the shoal at a uniform depth below sea level and prevents it from transitioning to an intertidal environment. Consequently, the long-term sedimentation rate approximates the rate of sea-level rise in the lower estuary (1-3 mm/yr). After the mid 1800s, the natural geometry of the lower Hudson

  13. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    Science.gov (United States)

    Drenth, Joost P H; Waxman, Stephen G

    2007-12-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Na(v)1.7 have been shown to cause primary erythermalgia and paroxysmal extreme pain disorder, while nonsense mutations in Na(v)1.7 result in loss of Na(v)1.7 function and a condition known as channelopathy-associated insensitivity to pain, a rare disorder in which affected individuals are unable to feel physical pain. This review highlights these recent developments and discusses the critical role of Na(v)1.7 in pain sensation in humans.

  14. Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome

    Directory of Open Access Journals (Sweden)

    Yiangou Yiangos

    2010-06-01

    Full Text Available Abstract Background Voltage gated sodium channels Nav1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders. Aims and Objectives To study Nav1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS, considered a neuropathic orofacial pain disorder. Methods Two groups of patients were recruited for this study. One group consisted of patients with dental pulpitis pain (n = 5 and controls (n = 12, and the other patients with BMS (n = 7 and controls (n = 10. BMS patients were diagnosed according to the International Association for the Study of Pain criteria; a pain history was collected, including the visual analogue scale (VAS. Immunohistochemistry with visual intensity and computer image analysis were used to evaluate levels of Nav1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group. Results There was a significantly increased visual intensity score for Nav1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Nav1.7 immunoreactive % area in the painful pulp group, but this was not statistically significant. When expressed as a ratio of the neurofilament % area, there was a strong trend for an increase of Nav1.7 in the painful pulp group. Nav1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls. Conclusion Nav1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Nav1.7 channel blockers should prioritise dental pulp pain rather than BMS.

  15. Assignment of human G-protein-coupled inward rectifier K{sup +} channel homolog GIRK3 gene to chromosome 1q21-q23

    Energy Technology Data Exchange (ETDEWEB)

    Lesage, F.; Fink, M.; Barhanin, J. [CNRS, Valbonne (France)] [and others

    1995-10-10

    More than 20 genes that encode voltage-gated and Ca{sup 2+}-dependent K{sup +} channels have been identified. These channels are involved in a wide variety of biological functions such as neuronal and muscle excitability, hormone secretion, and osmotic regulation. Two voltage-gated K{sup +} channel genes, KCNA1 and HERG, have been related to neurological and cardiac inherited disorders in humans. Missense mutations in the KCNA1 gene lead to episodic ataxia/myokimia syndrome. Missense, splice donor, and deletion mutations in the HERG gene have been shown to cause long QT syndrome. These two channels belong to the superfamily of cationic channels, which share the characteristic structural features of six transmembrane domains and one segment (called 115) involved in pore formation. 17 refs., 1 fig.

  16. Difference of Sodium Currents between Pediatric and Adult Human Atrial Myocytes: Evidence for Developmental Changes of Sodium Channels

    Directory of Open Access Journals (Sweden)

    Benzhi Cai, Xiaoqin Mu, Dongmei Gong, Shulin Jiang, Jianping Li, Qingxin Meng, Yunlong Bai, Yanju Liu, Xinyue Wang, Xueying Tan, Baofeng Yang, Yanjie Lu

    2011-01-01

    Full Text Available Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.

  17. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    Directory of Open Access Journals (Sweden)

    Acuña-Macías I

    2015-10-01

    Full Text Available Isabel Acuña-Macías,1 Eunice Vera,1 Alma Yolanda Vázquez-Sánchez,1 María Eugenia Mendoza-Garrido,2 Javier Camacho1 1Department of Pharmacology, 2Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico Abstract: Oncogenic ether à-go-go-1 (Eag1 potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. Keywords: lung cancer, serum deprivation, ether à-go-go, potassium channels, EGF, epidermal growth factor, ERK 1/2

  18. Comparison of Channelized Hotelling and Human Observers inDetermining Optimum OS-EM Reconstruction Parameters for MyocardialSPECT

    Energy Technology Data Exchange (ETDEWEB)

    Gilland, Karen L.; Tsui, Benjamin M.W.; Qi, Yujin; Gullberg,Grant T.

    2005-07-01

    The performance of the Channelized Hotelling Observer (CHO)was compared to that of human observers for determining optimumparameters for the iterative OS-EM image reconstruction method for thetask of defect detection in myocardial SPECT images. The optimumparameters were those that maximized defect detectability in the SPECTimages. Low noise, parallel SPECT projection data, with and without ananterior, inferior or lateral LV wall defect, were simulated using theMonte Carlo method. Poisson noise was added to generate noisyrealizations. Data were reconstructed using OS-EM at 1&4subsets/iteration and at 1, 3, 5, 7&9 iterations. Images wereconverted to 2D short-axis slices with integer pixel values. The CHO used3 radially-symmetric, 2D channels, with varying levels of internalobserver noise. For each parameter setting, 600 defect-present and 600defect-absent image vectors were used to calculate the detectabilityindex (dA). The human observers rated the likelihood that a defect waspresent in a specified location. For each parameter setting, the AUC wasestimated from 48 defect-present and 48 defect-absent images. Thecombined human observer results showed the optimum parameter settingcould be in the range 5-36 updates ([number of subsets]/iteration enumber of iterations). The CHO results showed the optimum parametersetting to be 4-5 updates. The performance of the CHO was much moresensitive to the reconstruction parameter setting than was that of thehuman observers. The rankings of the CHO detectability values did notchange with varying levels of internal noise.

  19. The Inhibition by Oxaliplatin, a Platinum-Based Anti-Neoplastic Agent, of the Activity of Intermediate-Conductance Ca2+-Activated K+ Channels in Human Glioma Cells

    Directory of Open Access Journals (Sweden)

    Mei-Han Huang

    2015-10-01

    Full Text Available Oxaliplatin (OXAL is a third-generation organoplatinum which is effective against advanced cancer cells including glioma cells. How this agent and other related compounds interacts with ion channels in glioma cells is poorly understood. OXAL (100 µM suppressed the amplitude of whole-cell K+ currents (IK; and, either DCEBIO or ionomycin significantly reversed OXAL-mediated inhibition of IK in human 13-06-MG glioma cells. In OXAL-treated cells, TRAM-34 did not suppress IK amplitude in these cells. The intermediate-conductance Ca2+-activated K+ (IKCa channels subject to activation by DCEBIO and to inhibition by TRAM-34 or clotrimazole were functionally expressed in these cells. Unlike cisplatin, OXAL decreased the probability of IKCa-channel openings in a concentration-dependent manner with an IC50 value of 67 µM. No significant change in single-channel conductance of IKCa channels in the presence of OXAL was demonstrated. Neither large-conductance Ca2+-activated K+ channels nor inwardly rectifying K+ currents in these cells were affected in the presence of OXAL. OXAL also suppressed the proliferation and migration of 13-06-MG cells in a concentration- and time-dependent manner. OXAL reduced IKCa-channel activity in LoVo colorectal cancer cells. Taken together, the inhibition by OXAL of IKCa channels would conceivably be an important mechanism through which it acts on the functional activities of glioma cells occurring in vivo.

  20. The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK.

    Science.gov (United States)

    Beltrán, Leopoldo R; Dawid, Corinna; Beltrán, Madeline; Gisselmann, Guenter; Degenhardt, Katharina; Mathie, Klaus; Hofmann, Thomas; Hatt, Hanns

    2013-01-01

    For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P) potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1), TASK-3 (K2P 9.1), and TRESK (K2P 18.1) channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreased the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

  1. The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK

    Directory of Open Access Journals (Sweden)

    Leopoldo Raul Beltran

    2013-11-01

    Full Text Available For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1, TASK-3 (K2P 9.1, and TRESK (K2P 18.1 channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreases the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

  2. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

    Science.gov (United States)

    Marie, Chelsea; Verkerke, Hans P; Theodorescu, Dan; Petri, William A

    2015-09-08

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

  3. Biophysical assessment of human aquaporin-7 as a water and glycerol channel in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Ana Madeira

    Full Text Available The plasma membrane aquaporin-7 (AQP7 has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.

  4. Implications of Human Transient Receptor Potential Melastatin 8 (TRPM8) Channel Gating from Menthol Binding Studies of the Sensing Domain.

    Science.gov (United States)

    Rath, Parthasarathi; Hilton, Jacob K; Sisco, Nicholas J; Van Horn, Wade D

    2016-01-12

    The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary cold sensor in humans. TRPM8 is gated by physiologically relevant cold temperatures and chemical ligands that induce cold sensations, such as the analgesic compound menthol. Characterization of TRPM8 ligand-gated channel activation will lead to a better understanding of the fundamental mechanisms that underlie TRPM8 function. Here, the direct binding of menthol to the isolated hTRPM8 sensing domain (transmembrane helices S1-S4) is investigated. These data are compared with two mutant sensing domain proteins, Y745H (S2 helix) and R842H (S4 helix), which have been previously identified in full length TRPM8 to be menthol insensitive. The data presented herein show that menthol specifically binds to the wild type, Y745H, and R842H TRPM8 sensing domain proteins. These results are the first to show that menthol directly binds to the TRPM8 sensing domain and indicates that Y745 and R842 residues, previously identified in functional studies as crucial to menthol sensitivity, do not affect menthol binding but instead alter coupling between the sensing domain and the pore domain.

  5. Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa

    Science.gov (United States)

    Hong, Hee-Kyung; Yoon, Weon-Jong; Kim, Young Ho; Yoo, Eun-Sook

    2009-01-01

    Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (IHERG) and the tail currents of HERG (Itail). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both IHERG and Itail were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either IHERG or Itail. The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 µg/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of Itail progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect. PMID:19949665

  6. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  7. Potent suppression of Kv1.3 potassium channel and IL-2 secretion by diphenyl phosphine oxide-1 in human T cells.

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    Full Text Available Diphenyl phosphine oxide-1 (DPO-1 is a potent Kv1.5 channel inhibitor that has therapeutic potential for the treatment of atrial fibrillation. Many other Kv1.5 channel blockers also potently inhibit the Kv1.3 channel, but whether DPO-1 blocks Kv1.3 channels has not been investigated. The Kv1.3 channel is highly expressed in activated T cells, which is considered a favorable target for immunomodulation. Accordingly, we hypothesized that DPO-1 may exert immunosuppressive and anti-inflammatory effects by inhibiting Kv1.3 channel activity. In this study, DPO-1 blocked Kv1.3 current in a voltage-dependent and concentration-dependent manner, with IC₅₀ values of 2.58 µM in Jurkat cells and 3.11 µM in human peripheral blood T cells. DPO-1 also accelerated the inactivation rate and negatively shifted steady-state inactivation. Moreover, DPO-1 at 3 µM had no apparent effect on the Ca²⁺ activated potassium channel (K(Ca current in both Jurkat cells and human peripheral blood T cells. In Jurkat cells, pre-treatment with DPO-1 for 24 h decreased Kv1.3 current density, and protein expression by 48±6% and 60±9%, at 3 and 10 µM, respectively (both p<0.05. In addition, Ca²⁺ influx to Ca²⁺-depleted cells was blunted and IL-2 production was also reduced in activated Jurkat cells. IL-2 secretion was also inhibited by the Kv1.3 inhibitors margatoxin and charybdotoxin. Our results demonstrate for the first time that that DPO-1, at clinically relevant concentrations, blocks Kv1.3 channels, decreases Kv1.3 channel expression and suppresses IL-2 secretion. Therefore, DPO-1 may be a useful treatment strategy for immunologic disorders.

  8. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    hypothesized that proteolytic processing of gammaENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and m...... higher abundance of full-length and furin-cleaved gammaENaC, with no significant change in the furin-cleaved-to-full-length gammaENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length gammaENaC by mAbinhibit. Prostasin/kallikrein-cleaved gamma......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  9. Multi-channel photon migration study in visible Chinese human muscle for optical detection of deep vein thrombosis

    Science.gov (United States)

    Sun, Yunlong; Li, Ting

    2016-03-01

    Deep vein thrombosis (DVT) always induced venous thrombosis. Most cases of venous thrombosis were induced by deep vein thrombosis (DVT), with high incidence rate of >60% in >60 years old people. Near-infrared spectroscopy (NIRS) were reported recently to be an intriguing and potential technique in detecting DVT in clinics. However, the photon transport is still unclear, which is crucial for the image reconstruction of the updated development called as NIRS-based DVT imager. Here we employed the Monte Carlo simulation software for 3D voxelized media (MCVM) and the Visible Chinese Human (VCH) model, which segmentation is finest in the world, to simulate multi-channel photon migration in calf muscle. And the image reconstruction of DVT hemodynamic distribution was achieved. This study, for the first time, provides the most realistic 3-D multichannel photon migration for NIRS study on DVT, and explored the image reconstruction for furtherly developing a NIRS-based DVT imager.

  10. Regulation of the human ether-a-go-go-related gene (hERG) potassium channel by Nedd4 family interacting proteins (Ndfips).

    Science.gov (United States)

    Kang, Yudi; Guo, Jun; Yang, Tonghua; Li, Wentao; Zhang, Shetuan

    2015-11-15

    The cardiac electrical disorder long QT syndrome (LQTS) pre-disposes affected individuals to ventricular arrhythmias and sudden death. Dysfunction of the human ether-a-go-go-related gene (hERG)-encoded rapidly activating delayed rectifier K(+) channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by anterograde trafficking of newly synthesized channels to and retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin (Ub) ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2) targets the PY motif of hERG channels to initiate channel degradation. Although both immature and mature hERG channels contain the PY motif, Nedd4-2 selectively mediates the degradation of mature hERG channels. In the present study, we demonstrate that Nedd4-2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Nedd4 family-interacting protein 1 (Ndfip1) and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4-2 to mediate the degradation of mature hERG proteins during channel trafficking to the plasma membrane. Although Ndfip2 directs Nedd4-2 to the Golgi apparatus, it also recruits Nedd4-2 to the multivesicular bodies (MVBs), which may impair MVB function and impede the degradation of mature hERG proteins mediated by Nedd4-2. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.

  11. Channel-opening kinetic mechanism for human wild-type GluK2 and the M867I mutant kainate receptor.

    Science.gov (United States)

    Han, Yan; Wang, Congzhou; Park, Jae Seon; Niu, Li

    2010-11-02

    GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms

  12. Identification and characterization of human neuronal voltage-gated calcium channel gamma 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By homologous expressed sequence tag (EST) searching,one EST (GenBank: W29095) was obtained,which shows 75% identity in 435 bp overlap with the coding sequence of mouse Cacng2 gene. A 1 545 bp cDNA fragment was obtained from the nested polymerase chain reaction (PCR) and rapid applification of cDNA end (RACE) reaction in the human brain prefrontal cortex cDNA library and the human brain Ready cDNA with the primers designed on W29095. The fragment contained a 948-bp open reading frame (ORF) encoding 315 amino acids,and was named CACNG3. As it was identical to a BAC clone (GenBank: AC004125) from chromosome 16p12-p13.1,the CACNG3 gene was mapped to human chromosome 16p12-p13.1,and the coding region was composed of 4 exons. Reverse transcription PCR (RT-PCR) analysis showed that the CACNG3 gene expressed in human adult brain and fetal brain. Single strand comformation polymorphism (SSCP) analysis was performed in 3 pedigrees with autosomal recessive retinitis pigmentosa,8 pedigrees with autosomal recessive retinitis pigmentosa accompanied by deafness and 2 pedigrees with epilepsy,but no mutation was detected.

  13. Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

    Directory of Open Access Journals (Sweden)

    Uttio Roy Chowdhury

    Full Text Available Elevated intraocular pressure (IOP is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001 when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89. In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002. Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/- mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm's canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

  14. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  15. Role in fast inactivation of conserved amino acids in the IV/S4-S5 loop of the human muscle Na+ channel.

    Science.gov (United States)

    Mitrovic, N; Lerche, H; Heine, R; Fleischhauer, R; Pika-Hartlaub, U; Hartlaub, U; George, A L; Lehmann-Horn, F

    1996-08-16

    Since it has been shown that point mutations in the S4-S5 loop of the Shaker K+ channel may disrupt fast inactivation, we investigated the role of three conserved amino acids in IV/S4-S5 of the adult human muscle Na+ channel (L1471, S1478, L1482). In contrast to the K+ channel mutations, the analogous substitutions in the Na+ channel (S1478A/C, L1482A) did not substantially affect fast inactivation. Nevertheless, the mutations S1478A/C/Q shifted the voltage dependence of steady-state inactivation; L1471Q and S1478C slowed recovery from inactivation. In contrast, a novel non-conserved IV/S4-S5 mutation causing paramyotonia congenita (F1473S) slowed fast inactivation 2-fold and accelerated recovery from inactivation 5-fold. The results indicate involvement of the IV/ S4-S5 loop of the human muscle Na+ channel in fast inactivation, but different roles for conserved amino acids among Na+ and K+ channels.

  16. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

    Directory of Open Access Journals (Sweden)

    Vonderlin N

    2015-02-01

    Full Text Available Nadine Vonderlin,1 Fathima Fischer,1 Edgar Zitron,1,2 Claudia Seyler,1 Daniel Scherer,1 Dierk Thomas,1,2 Hugo A Katus,1,2 Eberhard P Scholz1 1Department of Internal Medicine III, University Hospital Heidelberg, 2German Centre for Cardiovascular Research, Partner Site Heidelberg/Mannheim, Heidelberg, Germany Abstract: Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 µM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 µM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam. Keywords: midazolam, anesthetics, human ether

  17. [Three-dimensional structure of human Kv10.2 ion channel studied by single particle electron microscopy and molecular modeling].

    Science.gov (United States)

    Sokolova, O S; Shaĭtan, K V; Grizel', A V; Popinako, A V; Karlova, M G; Kirpichnikov, M P

    2012-01-01

    Here we present a three-dimensional structure of human voltage gated Kv10.2 ion channel solved at 2.5 nm resolution. We demonstrated that Kv10.2 channel structure is subdivided into two layers. For interpretation of the structure we used the homology modeling, using the transmembrane regions of MlotiK1 channel (C subunit), and cytoplasmic PAS-PAC and cNBD domains of the N-terminal tail of hERG (A subunit) and the bacterial cyclic nucleotide-activated K+ channel binding domain as the templates. The homologous transmembrane part can be fitted into the upper part of the reconstruction. The cytoplasmic domains form the structure, similar to a "hanging gondola", which is connected to the membrane-embedded domain with linkers. The length of linkers allow contacts between C-terminal cNBD domains and N-terminal PAS domains.

  18. Activation of human ether-a-go-go-related gene potassium channels by the diphenylurea 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643)

    DEFF Research Database (Denmark)

    Hansen, Rie Schultz; Diness, Thomas Goldin; Christ, Torsten

    2005-01-01

    The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased...... repolarization current could be a new antiarrhythmic principle, because it possibly would attenuate afterdepolarizations, ischemic leak currents, and reentry phenomena. Repolarization of the cardiac myocytes is crucially dependent on the late rapid delayed rectifier current (I(Kr)) conducted by ether......-a-go-go-related gene (ERG) potassium channels. We have developed the diphenylurea compound 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and tested whether this small organic molecule could increase the activity of human ERG (HERG) channels expressed heterologously. In Xenopus laevis oocytes, NS1643...

  19. Dielectrophoretic manipulation of human chromosomes in microfluidic channels: extracting chromosome dielectric properties

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Dimaki, Maria; Buckley, Sonia;

    2011-01-01

    An investigation of the dielectric properties of polyamine buffer prepared human chromosomes is presented in this paper. Chromosomes prepared in this buffer are only a few micrometers in size and shaped roughly like spherical discs. Dielectrophoresis was therefore chosen as the method...... of manipulation combined with a custom designed microfluidic system containing the required electrodes for dielectrophoresis experiments. Our results show that although this system is presently not able to distinguish between the different chromosomes, it can provide average data for the dielectric properties...... of human chromosomes in polyamine buffer. These can then be used to optimize system designs for further characterization and even sorting. The experimental data from the dielectrophoretic manipulation were combined with theoretical calculations to extract a range of values for the permittivity...

  20. An attempt to model the human body as a communication channel.

    Science.gov (United States)

    Wegmueller, Marc Simon; Kuhn, Andreas; Froehlich, Juerg; Oberle, Michael; Felber, Norbert; Kuster, Niels; Fichtner, Wolfgang

    2007-10-01

    Using the human body as a transmission medium for electrical signals offers novel data communication in biomedical monitoring systems. In this paper, galvanic coupling is presented as a promising approach for wireless intra-body communication between on-body sensors. The human body is characterized as a transmission medium for electrical current by means of numerical simulations and measurements. Properties of dedicated tissue layers and geometrical body variations are investigated, and different electrodes are compared. The new intra-body communication technology has shown its feasibility in clinical trials. Excellent transmission was achieved between locations on the thorax with a typical signal-to-noise ratio (SNR) of 20 dB while the attenuation increased along the extremities.

  1. Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

    Science.gov (United States)

    Che, Hui; Li, Gang; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-11-15

    Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

  2. Dynamic management of multi-channel interfaces for human interactions with computer-based intelligent assistants

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, T.D. Jr.

    1989-01-01

    For complex man-machine tasks where multi-media interaction with computer-based assistants is appropriate, a portion of the assistant's intelligence must be devoted to managing its communication processes with the user. Since people often serve the role of assistants, the conventions of human communication provide a basis for designing the communication processes of the computer-based assistant. Human decision making for communication requires knowledge of the user's style, the task demands, and communication practices, and knowledge of the current situation. Decisions necessary for effective communication, when, how, and what to communicate, can be expressed using these knowledge sources. A system based on human communication rules was developed to manage the communication decisions of an intelligent assistant. The Dynamic Communication Management (DCM) system consists of four components, three models and a manager. The model of the user describes the user's communication preferences for different task situations. The model of the task is used to establish the user's current activity and to describe how communication should be conducted for this activity. The communication model provides the rules needed to make decisions: when to communicate the message, how to present the message to the user, and what information should be communicated. The Communication Manager controls and coordinates these models to conduct all communication with the user. Performance with DCM as the interface to a simulated Flexible Manufacturing System (FMS) control task was established to learn about the potential benefits of the concept.

  3. Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus.

    Science.gov (United States)

    Surges, Rainer; Brewster, Amy L; Bender, Roland A; Beck, Heinz; Feuerstein, Thomas J; Baram, Tallie Z

    2006-07-01

    The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

  4. Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

    OpenAIRE

    2015-01-01

    Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Nav1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of ...

  5. The 3.5 ångström X-ray structure of the human connexin26 gap junction channel is unlikely that of a fully open channel.

    Science.gov (United States)

    Zonta, Francesco; Polles, Guido; Sanasi, Maria Federica; Bortolozzi, Mario; Mammano, Fabio

    2013-02-27

    The permeability of gap junction channels to metabolites, and not simply to small inorganic ions, is likely to play an important role in development, physiology as well as in etiology of several diseases. Here, we combined dual patch clamp and fluorescence imaging techniques with molecular dynamics (MD) simulations to investigate the permeation of calcein, a relatively large fluorescent tracer (MW 622 Da) through homomeric gap junction channels formed by wild type human connexin26 (hCx26wt) protomers. Our experimental data indicate that the unitary flux of calcein driven by a 125 μM concentration difference is Jpore = 226 molecule/s per channel. In the light of Eyring transition state theory adapted for the liquid phase, this value corresponds to an energy barrier of ~20 kBT (where kB is the Boltzmann constant and T is absolute temperature). The barrier predicted by our MD simulations, based on the 3.5 Å X-ray structural model of the hCx26wt gap junction channel, is ~45 kBT. The main contributions to the energetics of calcein permeation originated from the interaction between the permeating molecule and the charged aminoacids lining the channel pore. Assigning a fake zero total charge to the calcein molecule yielded a value for the barrier height compatible with the experimental data. These results can be accounted for by two different (although not mutually exclusive) hypotheses: (1) the X-ray model of the hCx26wt gap junction channel is not representative of a fully open state; (2) post translational modifications affecting the hCx26wt protein in our expression system differed from the modifications undergone by the proteins in the conditions used to obtain the crystal structure. Hypothesis (1) is compatible with data indicating that, only 10% or less of the channels forming a gap junction plaque are in the open state, and therefore the averaging procedure intrinsic in the generation of the crystal structure data more closely reflects that of a closed

  6. Functional Expression of Voltage-Gated Sodium Channels Navl.5 in Human Breast Caner Cell Line MDA-MB-231

    Institute of Scientific and Technical Information of China (English)

    Rui GAO; Jing WANG; Yi SHEN; Ming LEI; Zehua WANG

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are known to be involved in the initiation and progression of many malignancies,and the different subtypes of VGSCs play important roles in the metastasis cascade of many tumors.This study investigated the functional expression of Nav 1.5 and its effect on invasion behavior of human breast cancer cell line MDA-MB-231.The mRNA and pro-tein expression of Navl.5 was detected by real time PCR,Western Blot and immunofluorescence.The effects of Navl.5 on cell proliferation,migration and invasion were respectively assessed by MTT and Transwell.The effects of Nav1.5 on the secretion of matrix metalloproteases (MMPs) by MDA-MB-231 were analyzed by RT-PCR.The over-expressed Navl.5 was present on the membrane of MDA-MB-231 cells.The invasion ability in vitro and the MMP-9 mRNA expression were respec-tively decreased to (47.82±0.53)% and (43.97±0.64)% (P<0.05) respectively in MDA-MB-231 cells treated with VGSCs specific inhibitor tetrodotoxin (TTX) by blocking Navl.5 activity.It was con-eluded that Nav1.5 functional expression potentiated the invasive behavior of human breast cancer cell line MDA-MB-231 by increasing the secretion of MMP-9.

  7. Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells.

    Science.gov (United States)

    Bomberger, Jennifer M; Coutermarsh, Bonita A; Barnaby, Roxanna L; Stanton, Bruce A

    2012-05-18

    Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

  8. Excitation-Contraction Coupling between Human Atrial Myocytes with Fibroblasts and Stretch Activated Channel Current: A Simulation Study

    Directory of Open Access Journals (Sweden)

    Heqing Zhan

    2013-01-01

    Full Text Available Myocytes have been regarded as the main objectives in most cardiac modeling studies and attracted a lot of attention. Connective tissue cells, such as fibroblasts (Fbs, also play crucial role in cardiac function. This study proposed an integrated myocyte-Isac-Fb electromechanical model to investigate the effect of Fbs and stretch activated ion channel current (Isac on cardiac electrical excitation conduction and mechanical contraction. At the cellular level, an active Fb model was coupled with a human atrial myocyte electrophysiological model (including Isac and a mechanical model. At the tissue level, electrical excitation conduction was coupled with an elastic mechanical model, in which finite difference method (FDM was used to solve the electrical excitation equations, while finite element method (FEM was used for the mechanics equations. The simulation results showed that Fbs and Isac coupling caused diverse effects on action potential morphology during repolarization, depolarized the resting membrane potential of the human atrial myocyte, slowed down wave propagation, and decreased strains in fibrotic tissue. This preliminary simulation study indicates that Fbs and Isac have important implications for modulating cardiac electromechanical behavior and should be considered in future cardiac modeling studies.

  9. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

    Science.gov (United States)

    Wu, Ye; Arai, Amy C; Rumbaugh, Gavin; Srivastava, Anand K; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E; Valle, David; Huganir, Richard L; Wang, Tao

    2007-11-13

    Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

  10. Assignment of the human gene for the water channel of renal collecting duct Aquaporin 2 (AQP2) to chromosome 12 region q12-->q13

    NARCIS (Netherlands)

    Deen, P M; Weghuis, D O; Sinke, R J; Geurts van Kessel, A; Wieringa, B; van Os, C H

    1994-01-01

    The chromosomal localization of the gene encoding Aquaporin 2 (previously called WCH-CD), which acts as a water channel in the collecting tubules of the kidney, was determined. Southern blot hybridizations of chromosomal DNA from a panel of 25 different human-rodent hybrid cell lines assigned AQP2 t

  11. COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

    National Research Council Canada - National Science Library

    Minho Kang; Gracious R. Ross; Hamid I. Akbarali

    2007-01-01

    ...) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCav1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite...

  12. COOH-terminal association of human smooth muscle calcium channel Ca^sub v^1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

    National Research Council Canada - National Science Library

    Minho Kang; Gracious R Ross; Hamid I Akbarali

    2007-01-01

    ...) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa...1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite...

  13. COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

    National Research Council Canada - National Science Library

    Kang, Minho; Ross, Gracious R; Akbarali, Hamid I

    2007-01-01

    ...) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite...

  14. 质粒shRNA体内干扰Klotho基因对窦房结通道基因的影响%Study on the Effect of Klotho Gene Interferred by Plasmid-mediated Short Hairpin RNA (shRNA) on Sinoatrial Node Pacing Channel Gene

    Institute of Scientific and Technical Information of China (English)

    蔡盈盈; 汪汉; 侯言彬; 房晨鹂; 田鹏; 王贵华; 李璐; 邓珏琳

    2013-01-01

    通过质粒shRNA体内干扰,研究Klotho基因与窦房结起搏通道相关基因HCN4及HCN2之间的关系,为病窦综合征的研究提供新思路.取C57BL/6J小鼠20只,分为4组,每组5只,分别为:质粒shRNA 24 h组、质粒shRNA 12 h组、生理盐水24 h组、生理盐水12h组.质粒shRNA组经尾静脉注射质粒shRNA 50 μL(1 μg质粒/μL),生理盐水组经尾静脉注射生理盐水50 μL.分别于注射12h及24 h后取窦房结周围组织,行RT-PCR检测各组小鼠的Klotho、HCN2、HCN4基因的mRNA水平.RT-PCR结果显示:与生理盐水12h组比较,shRNA 12 h组的klotho、HCN4和HCN2的mRNA表达量明显降低,均有统计学差异(P<0.05).以上结果提示,小鼠Klotho基因和窦房结起搏基因可能存在一定关系.%The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4and HCN2) for studying sick sinus syndrome,with Klotho gene under the interference of Plasmid-mediated short hairpin RNA.Twenty-five C57BL/6J mice were divided into four groups,i.e,plasmid shRNA 24h group,plasmid shRNA 12h group,sodium chloride 24h group and sodium chloride 12h group.Plasmid shRNA 50μL (1μg/μL) and sodium chloride 50μl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group.After 12h or 24h respectively,all mice were executed and their sinoatrial node tissues were cut.The mRNA of Klotho,HCN4 and HCN2 gene were detected by RT-PCR.The results of RT-PCR showed that Klotho,HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval.The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  15. Cyamemazine metabolites: effects on human cardiac ion channels in-vitro and on the QTc interval in guinea pigs.

    Science.gov (United States)

    Crumb, William; Benyamina, Amine; Arbus, Christophe; Thomas, George P; Garay, Ricardo P; Hameg, Ahcène

    2008-11-01

    Monodesmethyl cyamemazine and cyamemazine sulfoxide, the two main metabolites of the antipsychotic and anxiolytic phenothiazine cyamemazine, were investigated for their effects on the human ether-à-go-go related gene (hERG) channel expressed in HEK 293 cells and on native I(Na), I(Ca), I(to), I(sus) or I(K1) of human atrial myocytes. Additionally, cyamemazine metabolites were compared with terfenadine for their effects on the QT interval in anaesthetized guinea pigs. Monodesmethyl cyamemazine and cyamemazine sulfoxide reduced hERG current amplitude, with IC50 values of 0.70 and 1.53 microM, respectively. By contrast, at a concentration of 1 microM, cyamemazine metabolites failed to significantly affect I(Na), I(to), I(sus) or I(K1) current amplitudes. Cyamemazine sulfoxide had no effect on I(Ca) at 1 microM, while at this concentration, monodesmethyl cyamemazine only slightly (17%), albeit significantly, inhibited I(Ca) current. Finally, cyamemazine metabolites (5 mg kg(-1) i v.) were unable to significantly prolong QTc values in the guinea pig. Conversely, terfenadine (5 mg kg(-1) i.v.) significantly increased QTc values. In conclusion, cyamemazine metabolite concentrations required to inhibit hERG current substantially exceed those necessary to achieve therapeutic activity of the parent compound in humans. Moreover, cyamemazine metabolites, in contrast to terfenadine, do not delay cardiac repolarization in the anaesthetized guinea pig. These non-clinical findings explain the excellent cardiac safety records of cyamemazine during its 30 years of extensive therapeutic use.

  16. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  17. Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit.

    Science.gov (United States)

    Tan, Xiao-qiu; Cheng, Xiu-li; Yang, Yan; Yan, Li; Gu, Jing-li; Li, Hui; Zeng, Xiao-rong; Cao, Ji-min

    2014-11-01

    Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels. Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy. DS-201 (40-160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca(2+) and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity. DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.

  18. River channel sensitivity to change in the context of human activities and natural factors: an 80-year record of channel morphodynamics on the lower Santa Clara River, Ventura County, California

    Science.gov (United States)

    Downs, P. W.; Dusterhoff, S. R.; Sears, W. A.

    2010-12-01

    River channel adjustments arise from the application of numerous catchment-based stressors operating at different space and time scales. Natural stressors include the impact of climatic phenomena and their inheritance; human stressors include both direct and indirect factors whose impacts have grown in magnitude and intensity during the Anthropocene, especially since about 1945. Consequently, the sensitivity of river channel morphodynamics is likely to have changed also, with implications for landform understanding and river management. Reconstructing channel morphodynamics during the Anthropocene requires interpreting multiple historical and secondary data sources to document changes at sufficient (i.e., reach-scale) resolution: for the 60-km lower Santa Clara River (LSCR), Ventura County, California, we used flow, sediment and precipitation records, repeat aerial photographs, LiDAR data, repeat topographic surveys, in-channel vegetation data, field observations, numerical modeling of high flow events, and narrative accounts. The catchment historical context since European-American settlement includes periods dominated by ranching and colonization (ca.1820-1890), irrigations and diversions (ca.1890-1955), dams and river modifications (1955-1990), and urban population growth (1990-present). Natural stressors were investigated based on the correlation of instantaneous flood peaks with annual rainfall records in this semi-arid setting. Successful prediction of the majority of gauged floods since about 1950 allows a flood sequence to be reconstructed back to 1873. Floods are clustered and of considerably greater magnitude in El Nino years of the El Nino-Southern Oscillation. The great majority of sediment transport thus occurs in El Nino years so that the dominant discharge is the largest discharge on record, in contrast to humid-region alluvial rivers. Responding to these stressors, the average width of the active channel bed has become narrower by almost 50% (1938

  19. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

    Science.gov (United States)

    Brennan, Sarah C; Finney, Brenda A; Lazarou, Maria; Rosser, Anne E; Scherf, Caroline; Adriaensen, Dirk; Kemp, Paul J; Riccardi, Daniela

    2013-01-01

    Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match

  20. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

    Directory of Open Access Journals (Sweden)

    Sarah C Brennan

    Full Text Available Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E11.5-16.5 in mouse in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult. Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+ channels (VGCC, inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3, P/Q type (CaV2.1, N-type (CaV2.2, R-type (CaV2.3, and T-type (CaV3.2 and CaV3.3 VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3, demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to

  1. A contrast-sensitive channelized-Hotelling observer to predict human performance in a detection task using lumpy backgrounds and Gaussian signals

    Science.gov (United States)

    Park, Subok; Badano, Aldo; Gallas, Brandon D.; Myers, Kyle J.

    2007-03-01

    Previously, a non-prewhitening matched filter (NPWMF) incorporating a model for the contrast sensitivity of the human visual system was introduced for modeling human performance in detection tasks with different viewing angles and white-noise backgrounds by Badano et al. But NPWMF observers do not perform well detection tasks involving complex backgrounds since they do not account for random backgrounds. A channelized-Hotelling observer (CHO) using difference-of-Gaussians (DOG) channels has been shown to track human performance well in detection tasks using lumpy backgrounds. In this work, a CHO with DOG channels, incorporating the model of the human contrast sensitivity, was developed similarly. We call this new observer a contrast-sensitive CHO (CS-CHO). The Barten model was the basis of our human contrast sensitivity model. A scalar was multiplied to the Barten model and varied to control the thresholding effect of the contrast sensitivity on luminance-valued images and hence the performance-prediction ability of the CS-CHO. The performance of the CS-CHO was compared to the average human performance from the psychophysical study by Park et al., where the task was to detect a known Gaussian signal in non-Gaussian distributed lumpy backgrounds. Six different signal-intensity values were used in this study. We chose the free parameter of our model to match the mean human performance in the detection experiment at the strongest signal intensity. Then we compared the model to the human at five different signal-intensity values in order to see if the performance of the CS-CHO matched human performance. Our results indicate that the CS-CHO with the chosen scalar for the contrast sensitivity predicts human performance closely as a function of signal intensity.

  2. Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs.

    Science.gov (United States)

    Motin, Leonid; Durek, Thomas; Adams, David J

    2016-01-01

    Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Nav1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Nav1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na(+) current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na(+) current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.

  3. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Science.gov (United States)

    Zhang, Ying-Ying; Li, Gang; Che, Hui; Sun, Hai-Ying; Xiao, Guo-Sheng; Wang, Yan; Li, Gui-Rong

    2015-01-01

    Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  4. Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Ying-Ying Zhang

    Full Text Available Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa, a voltage-gated TTX-sensitive sodium current (INa.TTX, and an inward rectifier K+ current (IKir were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.

  5. The Small-Conductance Ca2+-Activated Potassium Channel, Subtype SK3, in the Human Myometrium Is Downregulated in Early Stages of Pregnancy

    Directory of Open Access Journals (Sweden)

    M. Rahbek

    2013-01-01

    Full Text Available The human myometrium is mainly relaxed during pregnancy, but, up to term, contractions become more coordinated and forceful in order to initiate delivery. Small conductance Ca2+-activated K+ channels (SK channels in human myometrium have been shown to be downregulated in late pregnancy. The aim was to investigate the presence of SK2 and SK3 in the human myometrium from nonpregnant women, pregnant women at term, and pregnancies delivered preterm and, in addition, to characterize the time of downregulation of these channels. Using qRT-PCR, we observed significantly lower levels of mRNA for SK2 than for SK3 in the nonpregnant tissue. The mRNA levels of SK3 were significantly reduced in tissue from pregnancies at term and pregnancies resulting in preterm deliveries, whereas no downregulation for SK2 was observed. Western blotting confirmed the qRT-PCR results. Using immunohistochemical staining, both SK2 and SK3 were detected in endometrial glandular epithelium. We conclude that SK3 mRNA is downregulated early in pregnancy—at least among those that result in preterm deliveries. Furthermore, we find that SK channels are expressed not only in the uterine smooth muscle but also in the endometrial epithelium.

  6. Combining cluster analysis, feature selection and multiple support vector machine models for the identification of human ether-a-go-go related gene channel blocking compounds.

    Science.gov (United States)

    Nisius, Britta; Göller, Andreas H; Bajorath, Jürgen

    2009-01-01

    Blockade of the human ether-a-go-go related gene potassium channel is regarded as a major cause of drug toxicity and associated with severe cardiac side-effects. A variety of in silico models have been reported to aid in the identification of compounds blocking the human ether-a-go-go related gene channel. Herein, we present a classification approach for the detection of diverse human ether-a-go-go related gene blockers that combines cluster analysis of training data, feature selection and support vector machine learning. Compound learning sets are first divided into clusters of similar molecules. For each cluster, independent support vector machine models are generated utilizing preselected MACCS structural keys as descriptors. These models are combined to predict human ether-a-go-go related gene inhibition of our large compound data set with consistent experimental measurements (i.e. only patch clamp measurements on mammalian cell lines). Our combined support vector machine model achieves a prediction accuracy of 85% on this data set and performs better than alternative methods used for comparison. We also find that structural keys selected on the basis of statistical criteria are associated with molecular substructures implicated in human ether-a-go-go related gene channel binding.

  7. Single-channel power meter for registration of human millimeter radiation

    Directory of Open Access Journals (Sweden)

    Ya. V. Savenko

    2013-07-01

    Full Text Available Introduction. The development of modern techniques for noninvasive diagnostic sets requirements to ensure complete diagnostic safety, efficiency, comprehensiveness while maintaining high reliability of quantitative results. One of the promising directions for creating such devices is based on the idea of using information properties of the extra low millimeter-wave radiation from the human body or other biological objects. Problem definition. The main difficulty in registration of such radiation is associated with exceptionally low power electromagnetic field generated by the body and the absence lack of opportunities for effective their reception by the radiation sensitive receivers. This is a significant issue of physical media relevant information signals that ensure effective processes in the body. Solutions to the problem. The absence of a necessary equipment for electromagnetic radiation registration of millimeter range extra low radiation until now makes impossible the diagnostics in this range. Power meter based on semiconductor sensors can improve the compactness of equipment, ease of use and efficiency in diagnostics. Description of the model and experiment. The receiver located on the surface of the skin can get a signal from the cell body. The information status of cells in the deeper layers of the body is transferred to the surface of cells and is a modulating factor for the generated millimeter radiation. Three basic hardware portions that make up its technical support should be included in the diagnostic system. There are antenna measuring unit and interface process unit, storage and presentation of information. These signals can be detected and processed by a computer. The range of information signals estimated spectral characteristics of low-frequency envelope of the received signal can be used as informative features. Application. Any disease of the body changes metabolic processes in cells, thereby initiating the process of

  8. BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo.

    Science.gov (United States)

    Lai, Michael H; Wu, Yuejin; Gao, Zhan; Anderson, Mark E; Dalziel, Julie E; Meredith, Andrea L

    2014-11-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1(-/-)) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1(-/-) SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate.

  9. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  10. Computerized image analysis of cell-cell interactions in human renal tissue by using multi-channel immunoflourescent confocal microscopy

    Science.gov (United States)

    Peng, Yahui; Jiang, Yulei; Liarski, Vladimir M.; Kaverina, Natalya; Clark, Marcus R.; Giger, Maryellen L.

    2012-03-01

    Analysis of interactions between B and T cells in tubulointerstitial inflammation is important for understanding human lupus nephritis. We developed a computer technique to perform this analysis, and compared it with manual analysis. Multi-channel immunoflourescent-microscopy images were acquired from 207 regions of interest in 40 renal tissue sections of 19 patients diagnosed with lupus nephritis. Fresh-frozen renal tissue sections were stained with combinations of immunoflourescent antibodies to membrane proteins and counter-stained with a cell nuclear marker. Manual delineation of the antibodies was considered as the reference standard. We first segmented cell nuclei and cell membrane markers, and then determined corresponding cell types based on the distances between cell nuclei and specific cell-membrane marker combinations. Subsequently, the distribution of the shortest distance from T cell nuclei to B cell nuclei was obtained and used as a surrogate indicator of cell-cell interactions. The computer and manual analyses results were concordant. The average absolute difference was 1.1+/-1.2% between the computer and manual analysis results in the number of cell-cell distances of 3 μm or less as a percentage of the total number of cell-cell distances. Our computerized analysis of cell-cell distances could be used as a surrogate for quantifying cell-cell interactions as either an automated and quantitative analysis or for independent confirmation of manual analysis.

  11. Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes.

    Science.gov (United States)

    Devi, Sulochana; Markandeya, Yogananda; Maddodi, Nityanand; Dhingra, Anuradha; Vardi, Noga; Balijepalli, Ravi C; Setaluri, Vijayasaradhi

    2013-05-01

    Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca(2+) uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca(2+) influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi /Go proteins, did not affect basal or mGluR6-induced Ca(2+) uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.

  12. The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress.

    Science.gov (United States)

    Hutnik, Cindy M L; Pocrnich, Cady E; Liu, Hong; Laird, Dale W; Shao, Qing

    2008-02-01

    To determine the role of connexin43 (Cx43) and gap junctional intercellular communication (GJIC) in the response of the human retinal pigment epithelial cell line ARPE-19 to oxidative stress. ARPE-19 cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH), and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays, and Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of ARPE-19 cells with shRNA vectors targeting Cx43 or vectors encoding Cx43, Cx26, and a disease-linked dominant negative Cx43 mutant (G21R) were used, and the effect on cell viability was assessed. t-BOOH-induced ARPE-19 cell death was correlated with reductions in GJIC and in the total level of Cx43 protein expression. Overexpression of Cx26 and Cx43 increased the viability of oxidant-treated ARPE-19 cells. Conversely, shRNA knockdown of Cx43, expression of a disease-linked dominant negative Cx43 mutant, and blocking GJIC with 18beta-glycyrrhetinic acid and flufenamic acid all increased t-BOOH-induced ARPE-19 cell death. Cx43-mediated protection of ARPE-19 cells from oxidative stress-induced death is dependent on functional Cx43 channels.

  13. Statistical epistasis and functional brain imaging support a role of voltage-gated potassium channels in human memory.

    Directory of Open Access Journals (Sweden)

    Angela Heck

    Full Text Available Despite the current progress in high-throughput, dense genome scans, a major portion of complex traits' heritability still remains unexplained, a phenomenon commonly termed "missing heritability." The negligence of analytical approaches accounting for gene-gene interaction effects, such as statistical epistasis, is probably central to this phenomenon. Here we performed a comprehensive two-way SNP interaction analysis of human episodic memory, which is a heritable complex trait, and focused on 120 genes known to show differential, memory-related expression patterns in rat hippocampus. Functional magnetic resonance imaging was also used to capture genotype-dependent differences in memory-related brain activity. A significant, episodic memory-related interaction between two markers located in potassium channel genes (KCNB2 and KCNH5 was observed (P(nominal combined=0.000001. The epistatic interaction was robust, as it was significant in a screening (P(nominal=0.0000012 and in a replication sample (P(nominal=0.01. Finally, we found genotype-dependent activity differences in the parahippocampal gyrus (P(nominal=0.001 supporting the behavioral genetics finding. Our results demonstrate the importance of analytical approaches that go beyond single marker statistics of complex traits.

  14. Calcitriol inhibits Ether-a go-go potassium channel expression and cell proliferation in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Becerra, Rocio [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Diaz, Lorenza, E-mail: lorenzadiaz@gmail.com [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Camacho, Javier [Department of Pharmacology, Centro de Investigacion y de Estudios Avanzados, Instituto Politecnico Nacional, Av. Instituto Politecnico Nacional 2508, San Pedro Zacatenco 07360, Mexico, D.F. (Mexico); Barrera, David; Ordaz-Rosado, David; Morales, Angelica [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Ortiz, Cindy Sharon [Department of Pathology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Avila, Euclides [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Bargallo, Enrique [Department of Breast Tumors, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Arrecillas, Myrna [Department of Pathology, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Halhali, Ali; Larrea, Fernando [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico)

    2010-02-01

    Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-a-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.

  15. A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.

    Science.gov (United States)

    Titus, Steven A; Beacham, Daniel; Shahane, Sampada A; Southall, Noel; Xia, Menghang; Huang, Ruili; Hooten, Elizabeth; Zhao, Yong; Shou, Louie; Austin, Christopher P; Zheng, Wei

    2009-11-01

    Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

  16. Cooperative gating between ion channels.

    Science.gov (United States)

    Choi, Kee-Hyun

    2014-01-01

    Cooperative gating between ion channels, i.e. the gating of one channel directly coupled to the gating of neighboring channels, has been observed in diverse channel types at the single-channel level. Positively coupled gating could enhance channel-mediated signaling while negative coupling may effectively reduce channel gating noise. Indeed, the physiological significance of cooperative channel gating in signal transduction has been recognized in several in vivo studies. Moreover, coupled gating of ion channels was reported to be associated with some human disease states. In this review, physiological roles for channel cooperativity and channel clustering observed in vitro and in vivo are introduced, and stimulation-induced channel clustering and direct channel cross linking are suggested as the physical mechanisms of channel assembly. Along with physical clustering, several molecular mechanisms proposed as the molecular basis for functional coupling of neighboring channels are covered: permeant ions as a channel coupling mediator, concerted channel activation through the membrane, and allosteric mechanisms. Also, single-channel analysis methods for cooperative gating such as the binomial analysis, the variance analysis, the conditional dwell time density analysis, and the maximum likelihood fitting analysis are reviewed and discussed.

  17. Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico); Sandoval, Alejandro [School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla (Mexico); Monroy, Alma; Felix, Ricardo [Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City (Mexico); Monjaraz, Eduardo, E-mail: emguzman@siu.buap.mx [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico)

    2010-12-03

    Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.

  18. S-carbocysteine-lysine salt monohydrate and cAMP cause non-additive activation of the cystic fibrosis transmembrane regulator channel in human respiratory epithelium.

    Science.gov (United States)

    Meyer, G; Doppierio, S; Daffonchio, L; Cremaschi, D

    1997-03-03

    S-Carbocysteine-lysine salt monohydrate (S-CMC-Lys) has been shown to open a Cl- channel in the trachea, thus aiding fluid secretion. The aim of this study was to characterize the channel and the action mechanism on a culture line of human respiratory epithelial cells. The patch-clamp technique (in cell-attached or inside-out configuration) and conventional micro-electrodes were used. The activity and density of a cAMP-dependent Cl- channel, identical to the cystic fibrosis transmembrane regulator (CFTR) channel, proved to be maximally stimulated by 100 microM S-CMC-Lys present in the cAMP-free cell incubation medium for 240-290 min (cell-attached configuration). Subsequent addition of cAMP to the medium did not determine any further activation. S-CMC-Lys acted mostly indirectly as, when placed in direct contact with a membrane patch, activation of the CFTR channel was nil (cytoplasmic side) or limited (external side).

  19. Effects of arsenic trioxide on voltage-dependent potassium channels and on cell proliferation of human multiple myeloma cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jin; WANG Wei; WEI Qing-fang; FENG Tie-ming; TAN Li-jun; YANG Bao-feng

    2007-01-01

    @@ Arsenic trioxide (ATO) can induce cellular apoptosis and inhibit the activities of multiple myeloma (MM)cells in vitro,1 but how it works is not very clear. Recent studies showed that ATO worked on the voltagedependent potassium channel and L-type calcium channel in myocardial cells,2-5 but the effect of ATO on ion channels of tumor cells was rarely reported. As the potassium channel plays an important role in controlling cell proliferation,6 we studied the effects of ATO on the voltage-dependent potassium current (Ikv) of the voltage-dependent potassium channel in an MM cell line,and probed into the relationship between changes of the Ikv caused by ATO and cell proliferation.

  20. Somatodendritic ion channel expression in substantia nigra pars compacta dopaminergic neurons across postnatal development.

    Science.gov (United States)

    Dufour, Martial A; Woodhouse, Adele; Goaillard, Jean-Marc

    2014-08-01

    Dopaminergic neurons of the substantia nigra pars compacta (SNc) are involved in the control of movement, sleep, reward, learning, and nervous system disorders and disease. To date, a thorough characterization of the ion channel phenotype of this important neuronal population is lacking. Using immunohistochemistry, we analyzed the somatodendritic expression of voltage-gated ion channel subunits that are involved in pacemaking activity in SNc dopaminergic neurons in 6-, 21-, and 40-day-old rats. Our results demonstrate that the same complement of somatodendritic ion channels is present in SNc dopaminergic neurons from P6 to P40. The major developmental changes were an increase in the dendritic range of the immunolabeling for the HCN, T-type calcium, Kv4.3, delayed rectifier, and SK channels. Our study sheds light on the ion channel subunits that contribute to the somatodendritic delayed rectifier (Kv1.3, Kv2.1, Kv3.2, Kv3.3), A-type (Kv4.3) and calcium-activated SK (SK1, SK2, SK3) potassium currents, IH (mainly HCN2, HCN4), and the L- (Cav1.2, Cav1.3) and T-type (mainly Cav3.1, Cav3.3) calcium currents in SNc dopaminergic neurons. Finally, no robust differences in voltage-gated ion channel immunolabeling were observed across the population of SNc dopaminergic neurons for each age examined, suggesting that differing levels of individual ion channels are unlikely to distinguish between specific subpopulations of SNc dopaminergic neurons. This is significant in light of previous studies suggesting that age- or region-associated variations in the expression profile of voltage-gated ion channels in SNc dopaminergic neurons may underlie their vulnerability to dysfunction and disease.

  1. Frequency- and state-dependent blockade of human ether-a-go-go-related gene K+ channel by arecoline hydrobromide.

    Science.gov (United States)

    Zhao, Xu-yan; Liu, Yu-qi; Fu, Yi-cheng; Xu, Bin; Gao, Jin-liao; Zheng, Xiao-qin; Lin, Min; Chen, Mei-yan; Li, Yang

    2012-03-01

    The rapidly activating delayed rectifier potassium current (I(Kr)), whose pore-forming alpha subunit is encoded by the human ether-a-go-go-related gene (hERG), is a key contributor to the third phase of action potential repolarization. The aim of this study was to investigate the effect and mechanism of arecoline hydrobromide induced inhibition of hERG K(+) current (I(hERG)). Transient transfection of hERG channel cDNA plasmid pcDNA3.1 into the cultured HEK293 cells was performed using Lipofectamine. A standard whole-cell patch-clamp technique was used to record the I(hERG) before and after the exposure to arecoline. Arecoline decreased the amplitude and the density of the I(hERG) in a concentration-dependent manner (IC(50) = 9.55 mmol/L). At test potential of +60 mV, the magnitude of I(hERG) tail at test pulse of -40 mV was reduced from (151.7 ± 6.2) pA/pF to (84.4 ± 7.6) pA/pF (P arecoline in the open and inactivated state was significant in a state-dependent manner. The maximal blockade was achieved in the inactivated state. Studies of gating mechanism showed that the steady-state activation curve of I(hERG) was significantly negatively shifted by arecoline. Time constants of activation were shortened. Steady-state inactivation curve and time constants of fast inactivation were not significantly affected by arecoline. Furthermore, the inhibition of I(hERG) by arecoline was characterized markedly by a frequency-dependent manner from 0.03 to 1.00 Hz pulse. Arecoline could potently block I(hERG) in both frequency and state-dependent manner.

  2. Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells

    OpenAIRE

    Park, Won Sun; Heo, Soon Chul; Jeon, Eun Su; Hong, Da Hye; Son, Youn Kyoung; Ko, Jae-Hong; Kim, Hyoung Kyu; Lee, Sun Young; Kim, Jae Ho; Han, Jin

    2013-01-01

    Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contra...

  3. From complex B(1) mapping to local SAR estimation for human brain MR imaging using multi-channel transceiver coil at 7T.

    Science.gov (United States)

    Zhang, Xiaotong; Schmitter, Sebastian; Van de Moortele, Pierre-Francois; Liu, Jiaen; He, Bin

    2013-06-01

    Elevated specific absorption rate (SAR) associated with increased main magnetic field strength remains a major safety concern in ultra-high-field (UHF) magnetic resonance imaging (MRI) applications. The calculation of local SAR requires the knowledge of the electric field induced by radio-frequency (RF) excitation, and the local electrical properties of tissues. Since electric field distribution cannot be directly mapped in conventional MR measurements, SAR estimation is usually performed using numerical model-based electromagnetic simulations which, however, are highly time consuming and cannot account for the specific anatomy and tissue properties of the subject undergoing a scan. In the present study, starting from the measurable RF magnetic fields (B1) in MRI, we conducted a series of mathematical deduction to estimate the local, voxel-wise and subject-specific SAR for each single coil element using a multi-channel transceiver array coil. We first evaluated the feasibility of this approach in numerical simulations including two different human head models. We further conducted experimental study in a physical phantom and in two human subjects at 7T using a multi-channel transceiver head coil. Accuracy of the results is discussed in the context of predicting local SAR in the human brain at UHF MRI using multi-channel RF transmission.

  4. The potential role of cobalt ions released from metal prosthesis on the inhibition of Hv1 proton channels and the decrease in Staphyloccocus epidermidis killing by human neutrophils.

    Science.gov (United States)

    Daou, Samira; El Chemaly, Antoun; Christofilopoulos, Panayiotis; Bernard, Louis; Hoffmeyer, Pierre; Demaurex, Nicolas

    2011-03-01

    Infection by Staphylococcus epidermidis is a devastating complication of metal-on-metal (MM) total hip arthroplasty (THA). Neutrophils are the first line of defense against infection, and these innate immune cells are potentially exposed to Co(2+) ions released in the peri-prosthetic tissue by the wear of MM THA. The toxicity of Co(2+) is still debated, but Co(2+) is a potential inhibitor of the Hv1 proton channel that sustains the production of superoxide by neutrophils. In this study, we show that the Co(2+) concentration in peri-prosthetic tissue from patients with MM THA averages 53 μM and that such high concentrations of Co(2+) alter the antibacterial activity of human neutrophils in vitro by inhibiting Hv1 proton channels. We show that submillimolar concentrations of Co(2+) inhibit proton currents, impair the extrusion of cytosolic acid, and decrease the production of superoxide in human neutrophils. As a result, Co(2+) reduces the ability of human neutrophils to kill two strains of Staphyloccocus epidermidis by up to 7-fold at the maximal concentration tested of 100 μM Co(2+). By inhibiting proton channels, the Co(2+) ions released by metal prostheses might therefore promote bacterial infections in patients with metal-on-metal total hip arthroplasty.

  5. Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

    Science.gov (United States)

    Perrin, Mark J; Kuchel, Philip W; Campbell, Terence J; Vandenberg, Jamie I

    2008-11-01

    Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

  6. The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.

    Science.gov (United States)

    Chen, Szu-Han; Fu, Ssu-Ju; Huang, Jing-Jia; Tang, Chih-Yung

    2016-01-18

    Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

  7. Human ether à-gogo K(+) channel 1 (hEag1) regulates MDA-MB-231 breast cancer cell migration through Orai1-dependent calcium entry.

    Science.gov (United States)

    Hammadi, Mehdi; Chopin, Valérie; Matifat, Fabrice; Dhennin-Duthille, Isabelle; Chasseraud, Maud; Sevestre, Henri; Ouadid-Ahidouch, Halima

    2012-12-01

    Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go-go (hEag1) K(+) channels as relevant player in controlling cell cycle and proliferation of non-invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA-MB-231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA-MB-231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca(2+) entry and MDA-MB-231 cell migration without affecting cell proliferation. Recent studies have reported that Ca(2+) entry through Orai1 channels is required for MDA-MB-231 cell migration. Down-regulation of hEag1 or Orai1 reduced Ca(2+) influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca(2+) influx or cell migration were observed in cells co-transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM(+)). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum-induced migration of BC cells by controlling the Ca(2+) entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development.

  8. Inhibition of human two-pore domain K+ channel TREK1 by local anesthetic lidocaine: negative cooperativity and half-of-sites saturation kinetics.

    Science.gov (United States)

    Nayak, Tapan K; Harinath, S; Nama, S; Somasundaram, K; Sikdar, S K

    2009-10-01

    TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h)TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC(50) value of 180 muM, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta119 C-terminal deletion mutant (hTREK1(wt)-Delta119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

  9. Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC

    Directory of Open Access Journals (Sweden)

    Galiñanes Manuel

    2002-07-01

    Full Text Available Abstract Background These studies investigate the role of mitoKATP channels, protein kinase C (PKC and Mitogen activated protein kinase (p38MAPK on the cardioprotection of ischemic (IP and pharmacological preconditioning (PP of the human myocardium and their sequence of activation. Results Right atrial appendages from patients undergoing elective cardiac surgery were equilibrated for 30 min and then subjected to 90 min of simulated ischemia followed by 120 min reoxygenation. At the end of each protocol creatinine kinase leakage (CK U/g wet wt and the reduction of MTT to formazan dye (mM/g wet wt were measured. Similar protection was obtained with α1 agonist phenylephrine, adenosine and IP and their combination did not afford additional cardioprotection. Blockade of mitoKATP channels with 5-hydroxydecanoate, PKC with chelerythrine, or p38MAPK with SB203580 abolished the protection of IP and of PP. In additional studies, the stimulation of mitoKATP channels with diazoxide or activation of PKC with PMA or p38MAPK with anisomycin induced identical protection to that of IP and PP. The protection induced by diazoxide was abolished by blockade of PKC and by blockade of p38MAPK. Furthermore, the protection induced by PMA was abolished by SB203580 but not by 5-hydroxydecanoate, whereas the protection induced by anisomycin was unaffected by either 5-hydroxydecanoate or chelerythrine. Conclusions Opening of mitoKATP channels and activation of PKC and p38MAPK are obligatory steps in the signal transduction cascade of IP and PP of the human myocardium with PKC activation being downstream of the opening of mitoKATP channels and upstream of p38MAPK activation.

  10. The First Extracellular Linker Is Important for Several Aspects of the Gating Mechanism of Human TRPA1 Channel

    Science.gov (United States)

    Marsakova, Lenka; Barvik, Ivan; Zima, Vlastimil; Zimova, Lucie; Vlachova, Viktorie

    2017-01-01

    Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel involved in pain, inflammation and itching. This channel gates in response to many irritant and proalgesic agents, and can be modulated by calcium and depolarizing voltage. While the closed-state structure of TRPA1 has been recently resolved, also having its open state is essential for understanding how this channel works. Here we use molecular dynamics simulations combined with electrophysiological measurements and systematic mutagenesis to predict and explore the conformational changes coupled to the expansion of the presumptive channel's lower gate. We show that, upon opening, the upper part of the sensor module approaches the pore domain of an adjacent subunit and the conformational dynamics of the first extracellular flexible loop may govern the voltage-dependence of multimodal gating, thereby serving to stabilize the open state of the channel. These results are generally important in understanding the structure and function of TRPA1 and offer new insights into the gating mechanism of TRPA1 and related channels. PMID:28197074

  11. Ion channels in toxicology.

    Science.gov (United States)

    Restrepo-Angulo, Iván; De Vizcaya-Ruiz, Andrea; Camacho, Javier

    2010-08-01

    Ion channels play essential roles in human physiology and toxicology. Cardiac contraction, neural transmission, temperature sensing, insulin release, regulation of apoptosis, cellular pH and oxidative stress, as well as detection of active compounds from chilli, are some of the processes in which ion channels have an important role. Regulation of ion channels by several chemicals including those found in air, water and soil represents an interesting potential link between environmental pollution and human diseases; for instance, de novo expression of ion channels in response to exposure to carcinogens is being considered as a potential tool for cancer diagnosis and therapy. Non-specific binding of several drugs to ion channels is responsible for a huge number of undesirable side-effects, and testing guidelines for several drugs now require ion channel screening for pharmaceutical safety. Animal toxins targeting human ion channels have serious effects on the population and have also provided a remarkable tool to study the molecular structure and function of ion channels. In this review, we will summarize the participation of ion channels in biological processes extensively used in toxicological studies, including cardiac function, apoptosis and cell proliferation. Major findings on the adverse effects of drugs on ion channels as well as the regulation of these proteins by different chemicals, including some pesticides, are also reviewed. Association of ion channels and toxicology in several biological processes strongly suggests these proteins to be excellent candidates to follow the toxic effects of xenobiotics, and as potential early indicators of life-threatening situations including chronic degenerative diseases.

  12. Targeted deletion of Kcne2 impairs HCN channel function in mouse thalamocortical circuits.

    Directory of Open Access Journals (Sweden)

    Shui-Wang Ying

    Full Text Available BACKGROUND: Hyperpolarization-activated, cyclic nucleotide-gated (HCN channels generate the pacemaking current, I(h, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of Kcne2 gene deletion on I(h properties and excitability in ventrobasal (VB and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2(+/+ and Kcne2(-/- mice. Kcne2 deletion shifted the voltage-dependence of I(h activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I(h density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4, although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2(-/- neurons. CONCLUSIONS/SIGNIFICANCE: Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically

  13. The CLC-2 Chloride Channel Modulates ECM Synthesis, Differentiation, and Migration of Human Conjunctival Fibroblasts via the PI3K/Akt Signaling Pathway.

    Science.gov (United States)

    Sun, Lixia; Dong, Yaru; Zhao, Jing; Yin, Yuan; Zheng, Yajuan

    2016-06-09

    Recent evidence suggests that chloride channels are critical for cell proliferation, migration, and differentiation. We examined the effects of transforming growth factor (TGF)-β1 on chloride channel expression and associations with human conjunctival fibroblast (HConF) biology. To investigate the potential role of chloride channel (CLC)-2 in migration, transition to myofibroblasts and extracellular matrix (ECM) synthesis of HconF, a small interfering RNA (siRNA) approach was applied. TGF-β1-induced migration and transition of fibroblasts to myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression, supported by increased endogenous expression of CLC-2 protein and mRNA transcripts. ECM (collagen I and fibronectin) synthesis in HConF was enhanced by TGF-β1. CLC-2 siRNA treatment reduced TGF-β1-induced cell migration, transition of fibroblasts to myofibroblasts, and ECM synthesis of HConF. CLC-2 siRNA treatment in the presence of TGF-β1 inhibited phosphorylation of PI3K and Akt in HConF. These findings demonstrate that CLC-2 chloride channels are important for TGF-β1-induced migration, differentiation, and ECM synthesis via PI3K/Akt signaling in HConF.

  14. The Inhibitory Effects of Ketamine on Human Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and Action Potential in Rabbit Sinoatrial Node.

    Science.gov (United States)

    Xing, Junlian; Zhang, Chi; Jiang, Wanzhen; Hao, Jie; Liu, Zhipei; Luo, Antao; Zhang, Peihua; Fan, Xinrong; Ma, Jihua

    2017-01-01

    To investigate the effects of ketamine on human hyperpolarization-activated cyclic nucleotide-gated (hHCN) 1, 2, 4 channel currents expressed in Xenopus oocytes and spontaneous action potentials (APs) of rabbit sinoatrial node (SAN). The 2-electrode voltage clamp and standard microelectrode techniques were respectively applied to record hHCN channels currents expressed in Xenopus oocytes and APs of SAN separated from rabbit heart. Ketamine (1-625 µmol/L) blocked hHCN1, 2, and 4 currents with IC50 of 67.0, 89.1, and 84.0 µmol/L, respectively, in a concentration-dependent manner. The currents were rapidly blocked by ketamine and partially recovered after washout. The steady-state activation curves of hHCN1, 2, and 4 currents demonstrated a concentration-dependent shift to the left and the rates of activation were significantly decelerated. But ketamine blocked hHCN channels in a voltage-independence and non-use-dependent manner, and did not modify the voltage dependence of activation and reversal potentials. Furthermore, ketamine suppressed phase-4 spontaneous depolarization rate in isolated rabbit SAN and decreased the beat rates in a concentration-dependent manner. Ketamine could inhibit hHCN channels expressed in Xenopus oocytes in a concentration-dependent manner as a close-state blocker and decrease beat rates of isolated rabbit SAN. This study may provide novel insights into other unexplained actions of ketamine. © 2017 S. Karger AG, Basel.

  15. Two mutations in the IV/S4-S5 segment of the human skeletal muscle Na+ channel disrupt fast and enhance slow inactivation.

    Science.gov (United States)

    Alekov, A K; Peter, W; Mitrovic, N; Lehmann-Horn, F; Lerche, H

    2001-06-29

    Fast and slow inactivation (FI, SI) of the voltage-gated Na+ channel are two kinetically distinct and structurally dissociated processes. The voltage sensor IV/S4 and the intracellular IV/S4-S5 loop have been shown to play an important role in FI mediating the coupling between activation and inactivation. Two mutations in IV/S4-S5 of the human muscle Na+ channel, L1482C/A, disrupt FI by inducing a persistent Na+ current, shifting steady-state inactivation in the depolarizing direction and accelerating its recovery. These effects were more pronounced for L1482A. In contrast, SI of L1482C/A channels was enhanced showing a more complete SI and a 3-fold slowing of its recovery. Effects on SI were more pronounced for L1482C. The results indicate an important role of the IV/S4-S5 loop not only in FI but also in SI of the Na+ channel.

  16. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    Science.gov (United States)

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  17. Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

    Science.gov (United States)

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

    2012-12-07

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

  18. The role of external Ca²⁺ in the action of Ca²⁺-channel agonists and antagonists on isolated human thoracic arteries.

    Science.gov (United States)

    Garaliene, V; Barsys, V; Giedraitis, S; Benetis, R; Krauze, A

    2014-02-01

    In systemic atherosclerosis develops the abnormal vascular tone which is associated with elevated calcium influx into smooth muscle cells and their calcification that may be proportional to the extent and severity of atherosclerotic disease. The goal of the present study was to investigate the responses of isolated human arterial samples to Ca²⁺-channel agonists and antagonists by varying the external Ca²⁺ concentration. Two dihydropyridine type calcium-channel blockers, amlodipine and cerebrocrast, were used in this study. The benzodiazepine-type calcium-channel blocker diltiazem, the benzimidazole derivative 1-acetyl-5,6-dimethoxy-2-methylthiobenzimidazole and 3,4'-bipyridine derivative milrinone were also used. Experiments were carried out on isolated human thoracic artery samples obtained from 74 patients, aged 38-88 years, during conventional myocardial revascularisation operations. The contraction of artery samples was recorded using an iFOT10 force transducer. Cumulative concentration-contraction curves of the tested agents (10⁻⁷ to 10⁻⁴ M) were established by varying the external Ca²⁺ concentration from 0.9 mM to 2.7 mM. Cerebrocrast, regardless of the Ca²⁺ concentration significantly increased arterial contraction, particularly at the lower Ca²⁺ (≈77%). Diltiazem, the benzimidazole derivative and milrinone caused the artery samples to relax at 10⁻⁴ M concentrations by 55%, 55% and 44%, respectively, when the external Ca²⁺ corresponded to the physiological standard. Shifting to lower or higher Ca²⁺ concentrations significantly altered the response of vessel samples by increasing their contraction. In conclusion, the present study shows that the response of isolated human thoracic artery samples to both the slow calcium channel suppressant diltiazem and to agonists of that channel (milrinone and the benzimidazole derivative) is regulated by the amount of calcium present in the physiological solution. Treatment with a slow

  19. CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype.

    Science.gov (United States)

    Fischer, Horst; Illek, Beate; Sachs, Lorne; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2010-10-01

    Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.

  20. K(v)7 channels: function, pharmacology and channel modulators.

    Science.gov (United States)

    Dalby-Brown, William; Hansen, Henrik H; Korsgaard, Mads P G; Mirza, Naheed; Olesen, Søren-P

    2006-01-01

    K(v)7 channels are unique among K(+) channels, since four out of the five channel subtypes have well-documented roles in the development of human diseases. They have distinct physiological functions in the heart and in the nervous system, which can be ascribed to their voltage-gating properties. The K(v)7 channels also lend themselves to pharmacological modulation, and synthetic openers as well as blockers of the channels, regulating neuronal excitability, have existed even before the K(v)7 channels were identified by cloning. In the present review we give an account on the focused efforts to develop selective modulators, openers as well as blockers, of the K(v)7 channel subtypes, which have been undertaken during recent years, along with a discussion of the K(v)7 ion channel physiology and therapeutic indications for modulators of the neuronal K(v)7 channels.

  1. Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

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    Uysal-Onganer Pinar

    2007-11-01

    Full Text Available Abstract Background Although a high level of functional voltage-gated sodium channel (VGSC expression has been found in strongly metastatic human and rat prostate cancer (PCa cells, the mechanism(s responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF, a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion. Results Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion. Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX suppressed the effect of EGF on all three metastatic cell behaviours studied. Conclusion 1 EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2 VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

  2. APETx1 from sea anemone Anthopleura elegantissima is a gating modifier peptide toxin of the human ether-a-go-go- related potassium channel.

    Science.gov (United States)

    Zhang, M; Liu, X-S; Diochot, S; Lazdunski, M; Tseng, G-N

    2007-08-01

    We studied the mechanism of action and the binding site of APETx1, a peptide toxin purified from sea anemone, on the human ether-a-go-go-related gene (hERG) channel. Similar to the effects of gating modifier toxins (hanatoxin and SGTx) on the voltage-gated potassium (Kv) 2.1 channel, APETx1 shifts the voltage-dependence of hERG activation in the positive direction and suppresses its current amplitudes elicited by strong depolarizing pulses that maximally activate the channels. The APETx1 binding site is distinctly different from that of a pore-blocking peptide toxin, BeKm-1. Mutations in the S3b region of hERG have dramatic impact on the responsiveness to APETx1: G514C potentiates whereas E518C abolishes the APETx1 effect. Restoring the negative charge at position 518 (methanethiosulfonate ethylsulfonate modification of 518C) partially restores APETx1 responsiveness, supporting an electrostatic interaction between E518 and APETx1. Among the three hERG isoforms, hERG1 and hERG3 are equally responsive to APETx1, whereas hERG2 is insensitive. The key feature seems to be an arginine residue uniquely present at the 514-equivalent position in hERG2, where the other two isoforms possess a glycine. Our data show that APETx1 is a gating modifier toxin of the hERG channel, and its binding site shares characteristics with those of gating modifier toxin binding sites on other Kv channels.

  3. The Effect of Trichostatin-A and Tumour Necrosis Factor on Expression of Splice Variants of the MaxiK and L-Type Channels in Human Myometrium

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    Sarah L. Waite

    2014-07-01

    Full Text Available The onset of human parturition is associated with up-regulation of pro-inflammatory cytokines including TNF as well as changes in ion flux, principally Ca2+ and K+, across the myometrial myocytes membrane. Elevation of intra-cellular Ca2+ from the sarcoplasmic reticulum opens L-type Ca2+ channels; in turn this increased calcium level activates MaxiK channels leading to relaxation. While the nature of how this cross-talk is governed remains unclear, our previous work demonstrated that the pro-inflammatory cytokine, TNF, and the histone deacetylase inhibitor, TSA, exerted opposing effects on the expression of the pro-quiescent Gs gene in human myometrial cells. Consequently, in this study we demonstrate that the different channel splice variants for both MaxiK and L-Type Ca2+ channel are expressed in primary myometrial myocytes. MaxiK mRNA expression was sensitive to TSA stimulation, this causing repression of the M1, M3 and M4 splice variants. A small but not statistically significantly increase in MaxiK expression was also seen in response to TNF. In contrast to this, expression of LTCC splice variants was seen to be influenced by both TNF and TSA: TNF induced overall increase in total LTCC expression while TSA stimulated a dual effect: causing induction of LTCC exon 8 expression but repressing expression of other LTCC splice variants including that encoding exons 30, 31, 33 & 34, exons 30-34 and exons 40-43. The significance of these observations is discussed herein.

  4. Evidence of specialized tissue in human interatrial septum: histological, immunohistochemical and ultrastructural findings.

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    Lubov B Mitrofanova

    Full Text Available There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS. The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO and flap valve.Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17--longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy.In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95% cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03 and AF history (P = 0.045. Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells.Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.

  5. Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes.

    Science.gov (United States)

    Kuno, M; Goronzy, J; Weyand, C M; Gardner, P

    Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.

  6. The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker.

    Directory of Open Access Journals (Sweden)

    Xiao-Fang Yang

    Full Text Available Selective blockade of Kv1.3 channels in effector memory T (T(EM cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3 extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314 was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1, Kv1.2(hKv1.2, Kv1.4(hKv1.4, Kv1.5(hKv1.5, KCa3.1(hKCa3.1, HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+ or voltage-gated Na(+ currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.

  7. Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

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    Zeineb Es-Salah-Lamoureux

    Full Text Available BACKGROUND: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. METHODS AND FINDINGS: Tetramethylrhodamine-5-maleimide (TMRM fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449 in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2 of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON, with V((1/2 (,1 = -37.8+/-1.7 mV, and V((1/2 (,2 = 43.5+/-7.9 mV. The first phase, V((1/2 (,1, was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2 = -18.3+/-1.2 mV, and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2 = -34.4+/-1.5 mV, suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2 = -20.6+/-1.2, k = 11.4 mV and L520C quenching during depolarization (V((1/2 = -26.8+/-1.0, k = 13.3 mV matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing. CONCLUSION: THE DATA INDICATE: 1

  8. Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference

    Science.gov (United States)

    Slowik, Daria; Henderson, Richard

    2015-01-01

    With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3 > hNaV1.2 > hNaV1.1 > hNaV1.6 > hNaV1.3 > hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel. PMID:25838126

  9. Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference.

    Science.gov (United States)

    Slowik, Daria; Henderson, Richard

    2015-07-01

    With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.

  10. Voltage-Activated Calcium Channels as Functional Markers of Mature Neurons in Human Olfactory Neuroepithelial Cells: Implications for the Study of Neurodevelopment in Neuropsychiatric Disorders

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    Héctor Solís-Chagoyán

    2016-06-01

    Full Text Available In adulthood, differentiation of precursor cells into neurons continues in several brain structures as well as in the olfactory neuroepithelium. Isolated precursors allow the study of the neurodevelopmental process in vitro. The aim of this work was to determine whether the expression of functional Voltage-Activated Ca2+ Channels (VACC is dependent on the neurodevelopmental stage in neuronal cells obtained from the human olfactory epithelium of a single healthy donor. The presence of channel-forming proteins in Olfactory Sensory Neurons (OSN was demonstrated by immunofluorescent labeling, and VACC functioning was assessed by microfluorometry and the patch-clamp technique. VACC were immunodetected only in OSN. Mature neurons responded to forskolin with a five-fold increase in Ca2+. By contrast, in precursor cells, a subtle response was observed. The involvement of VACC in the precursors’ response was discarded for the absence of transmembrane inward Ca2+ movement evoked by step depolarizations. Data suggest differential expression of VACC in neuronal cells depending on their developmental stage and also that the expression of these channels is acquired by OSN during maturation, to enable specialized functions such as ion movement triggered by membrane depolarization. The results support that VACC in OSN could be considered as a functional marker to study neurodevelopment.

  11. Voltage-gated sodium channels in taste bud cells

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    Williams Mark E

    2009-03-01

    Full Text Available Abstract Background Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. Results We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. Conclusion SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  12. The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein

    Science.gov (United States)

    Fucile, Sergio; Palma, Eleonora; Martínez-Torres, Ataúlfo; Miledi, Ricardo; Eusebi, Fabrizio

    2002-01-01

    Neuronal nicotinic acetylcholine (AcCho) receptors composed of α7-subunits (α7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) α7-AcChoRs, we identified two classes of channel conductance: a low conductance (γL) of 72 pS and a high one (γH) of 87 pS, with mean open-times (τop) of 0.6 ms. The same classes of conductances, but longer τop (3 ms), were seen in experiments with chimeric α7 receptors in which the wtα7 extracellular C terminus was fused to the green fluorescent protein (wtα7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing α7 receptors carrying a Leu-to-Thr 248 mutation (mutα7) or oocytes expressing chimeric mutα7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtα7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter τop, than those activated by AcCho. Accordingly, human homomeric wtα7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutα7 receptors open channels of lower conductance and longer lifetime than those gated by wtα7-AcChoRs, and these parameters are not greatly altered by fusing the mutα7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function. PMID:11891309

  13. Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin.

    Science.gov (United States)

    Thomas, Dierk; Wimmer, Anna-Britt; Wu, Kezhong; Hammerling, Bettina C; Ficker, Eckhard K; Kuryshev, Yuri A; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A

    2004-05-01

    Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG

  14. Evaluation of mental workload and familiarity in human computer interaction with integrated development environments using single-channel EEG

    OpenAIRE

    2015-01-01

    With modern developments in sensing technology it has become possible to detect and classify brain activity into distinct states such as attention and relaxation using commercially avail- able EEG devices. These devices provide a low-cost and minimally intrusive method to observe a subject’s cognitive load whilst interacting with a computer system, thus providing a basis for deter- mining the overall effectiveness of the design of a computer interface. In this paper, a single-channel dry sens...

  15. Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

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    Ovcaric Mick

    2005-09-01

    Full Text Available Abstract Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3' polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO. In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090. Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05. The prevalence of the long CAG repeat (>19 repeats did not reach statistical significance in migraineurs (P = 0.15, nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively, or between MA vs MO (P = 0.40. Conclusion This association study provides no evidence that length variations of the second polyglutamine array in

  16. Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts

    Science.gov (United States)

    Tajhya, Rajeev B; Hu, Xueyou; Tanner, Mark R; Huq, Redwan; Kongchan, Natee; Neilson, Joel R; Rodney, George G; Horrigan, Frank T; Timchenko, Lubov T; Beeton, Christine

    2016-01-01

    Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1. PMID:27763639

  17. Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane.

    Science.gov (United States)

    Witschas, Katja; Jobin, Marie-Lise; Korkut, Dursun Nizam; Vladan, Maria Magdalena; Salgado, Gilmar; Lecomte, Sophie; Vlachova, Viktorie; Alves, Isabel D

    2015-05-01

    The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.

  18. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  19. Functional expression of smooth muscle-specific ion channels in TGF-β(1)-treated human adipose-derived mesenchymal stem cells.

    Science.gov (United States)

    Park, Won Sun; Heo, Soon Chul; Jeon, Eun Su; Hong, Da Hye; Son, Youn Kyoung; Ko, Jae-Hong; Kim, Hyoung Kyu; Lee, Sun Young; Kim, Jae Ho; Han, Jin

    2013-08-15

    Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca(2+), big-conductance Ca(2+)-activated K(+) (BKCa), and voltage-dependent K(+) (Kv) currents in TGF-β1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.

  20. Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells

    Science.gov (United States)

    Park, Won Sun; Heo, Soon Chul; Jeon, Eun Su; Hong, Da Hye; Son, Youn Kyoung; Ko, Jae-Hong; Kim, Hyoung Kyu; Lee, Sun Young; Kim, Jae Ho

    2013-01-01

    Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca2+, big-conductance Ca2+-activated K+ (BKCa), and voltage-dependent K+ (Kv) currents in TGF-β1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs. PMID:23761629

  1. Quantification of the functional expression of the Ca(2+) -activated K(+) channel KCa 3.1 on microglia from adult human neocortical tissue

    DEFF Research Database (Denmark)

    Blomster, Linda V; Strøbaek, Dorte; Hougaard, Charlotte;

    2016-01-01

    at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither...... by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals...

  2. Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

    Science.gov (United States)

    Chen, Mao Xiang; Gorman, Shelby A; Benson, Bill; Singh, Kuljit; Hieble, J Paul; Michel, Martin C; Tate, Simon N; Trezise, Derek J

    2004-06-01

    The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

  3. Inhibition by a novel anti-arrhythmic agent, NIP-142, of cloned human cardiac K+ channel Kv1.5 current.

    Science.gov (United States)

    Matsuda, T; Masumiya, H; Tanaka, N; Yamashita, T; Tsuruzoe, N; Tanaka, Y; Tanaka, H; Shigenoba, K

    2001-03-16

    NIP-142 was shown to prolong atrial effective refractory period and to terminate atrial fibrillation and flutter in in vivo canine models. To obtain information on its antiarrhythmic action, we examined the effect of NIP-142 on cloned human cardiac K+ channel Kv1.5 (hKv1.5) currents stably expressed in a human cell line using whole-cell voltage clamp methods. NIP-142 inhibited the hKv1.5 current in a concentration-dependent and voltage-independent manner. The inhibition was larger at the end of depolarizing pulse than at the outward current peak. The IC50 for inhibition of the steady-state phase was 4.75 microM. A cross-over phenomenon was observed when current traces in the absence and presence of NIP-142 were superimposed. Inhibition of hKv1.5 current by NIP-142 was frequency-independent; changing the depolarizing pulse frequencies (0.1, 0.2, 1 Hz) and little effect on the degree of inhibition. NIP-142 decreased the maximal peak amplitude of kHv1.5 current at the first command pulse after 3 min rest in the presence of the drug. These results suggest that NIP-142 has inhibitory effects on the hKv 1.5 current through interaction with both open and closed states of the channel, which may underlie its antiarrhythmic activity in the atria.

  4. The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation

    DEFF Research Database (Denmark)

    Kaestner, Lars; Christophersen, Palle; Bernhardt, Ingolf;

    2000-01-01

    Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor......Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor...

  5. [Familial hyperkalemic periodic paralysis: a brief review of the adult human skeletal muscle sodium channel and the application of LA-PCR to the SCN4A gene analysis].

    Science.gov (United States)

    Sakoda, S; Nakagawa, M; Arimura, Y; Arimura, K; Osame, M

    1997-12-01

    Recent work has revealed that familial hyperkalemic periodic paralysis, paramyotonia congenita and other non-dystrophic myotonias result from point mutations in the gene encoding the alpha-subunit of the adult human skeletal muscle sodium channel (SCN4A). Sodium channel myotonias are a diverse group of skeletal muscle disorders that share a common pathophysiological mechanism: all are caused by impaired rapid inactivation of skeletal muscle sodium channel. Clinical studies, pharmacology, electrophysiology and molecular genetics have contributed to an elucidation of the genotype-phenotype correlation within these disorders. This article briefly reviews recent advances in our understanding of skeletal muscle sodium channel and sodium channel myotonias. The application of LA-PCR to the SCN4A gene analysis is also referred.

  6. Functional interaction between S1 and S4 segments in voltage-gated sodium channels revealed by human channelopathies.

    Science.gov (United States)

    Amarouch, Mohamed-Yassine; Kasimova, Marina A; Tarek, Mounir; Abriel, Hugues

    2014-01-01

    The p.I141V mutation of the voltage-gated sodium channel is associated with several clinical hyper-excitability phenotypes. To understand the structural bases of the p.I141V biophysical alterations, molecular dynamics simulations were performed. These simulations predicted that the p.I141V substitution induces the formation of a hydrogen bond between the Y168 residue of the S2 segment and the R225 residue of the S4 segment. We generated a p.I141V-Y168F double mutant for both the Nav1.4 and Nav1.5 channels. The double mutants demonstrated the abolition of the functional effects of the p.I141V mutation, consistent with the formation of a specific interaction between Y168-S2 and R225-S4. The single p.Y168F mutation, however, positively shifted the activation curve, suggesting a compensatory role of these residues on the stability of the voltage-sensing domain.

  7. COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation.

    Science.gov (United States)

    Kang, Minho; Ross, Gracious R; Akbarali, Hamid I

    2007-12-01

    The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

  8. Role in fast inactivation of the IV/S4-S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis.

    Science.gov (United States)

    Lerche, H; Peter, W; Fleischhauer, R; Pika-Hartlaub, U; Malina, T; Mitrovic, N; Lehmann-Horn, F

    1997-12-01

    1. In order to investigate the role in fast inactivation of the cytoplasmic S4-S5 loop of the fourth domain (IV/S4-S5) within the alpha-subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole-cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated. 2. Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2- and 1.5-fold, respectively, and L1482C induced a steady-state Na+ current (Iss) of 3% of peak current (Ipeak) (1% for wild-type). 3. Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9-fold slowing of inactivation; Iss/Ipeak, 3.6%; +15 mV shift in steady-state inactivation; 2- to 3-fold acceleration of recovery from inactivation), A1481C (3-fold; 14%; +20 mV; no change) and F1473C (2.5-fold; 2.4%; +8 mV; 1.5-fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20-30 mV hyperpolarizing shift of the inactivation curve associated with a 3- to 4-fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed. 4. We conclude that the IV/S4-S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative

  9. IgG and complement deposition and neuronal loss in cats and humans with epilepsy and voltage-gated potassium channel complex antibodies.

    Science.gov (United States)

    Klang, Andrea; Schmidt, Peter; Kneissl, Sibylle; Bagó, Zoltán; Vincent, Angela; Lang, Bethan; Moloney, Teresa; Bien, Christian G; Halász, Péter; Bauer, Jan; Pákozdy, Akos

    2014-05-01

    Voltage-gated potassium channel complex (VGKC-complex) antibody (Ab) encephalitis is a well-recognized form of limbic encephalitis in humans, usually occurring in the absence of an underlying tumor. The patients have a subacute onset of seizures, magnetic resonance imaging findings suggestive of hippocampal inflammation, and high serum titers of Abs against proteins of the VGKC-complex, particularly leucine-rich, glioma-inactivated 1 (LGI1). Most patients are diagnosed promptly and recover substantially with immunotherapies; consequently, neuropathological data are limited. We have recently shown that feline complex partial cluster seizures with orofacial involvement (FEPSO) in cats can also be associated with Abs against VGKC-complexes/LGI1. Here we examined the brains of cats with FEPSO and compared the neuropathological findings with those in a human with VGKC-complex-Ab limbic encephalitis. Similar to humans, cats with VGKC-complex-Ab and FEPSO have hippocampal lesions with only moderate T-cell infiltrates but with marked IgG infiltration and complement C9neo deposition on hippocampal neurons, associated with neuronal loss. These findings provide further evidence that FEPSO is a feline form of VGKC-complex-Ab limbic encephalitis and provide a model for increasing understanding of the human disease.

  10. Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts

    Science.gov (United States)

    Fedorov, Vadim V.; Glukhov, Alexey V.; Ambrosi, Christina M.; Kostecki, Geran; Chang, Roger; Janks, Deborah; Schuessler, Richard B.; Moazami, Nader; Nichols, Colin G.; Efimov, Igor R.

    2011-01-01

    INTRODUCTION This study compared the effects of ATP-regulated potassium channel (KATP) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. METHODS We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial-ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the KATP targets Kir6.1, Kir6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. RESULTS In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by −21±3% and −27±13%, p<0.01) and ventricles (by −28±7% and −28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (−46 to - 80%, p<0.01) and ventricles (−65 to −93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1 Hz and 17.5 ±5.1 Hz, respectively. Glibenclamide (10 μM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of KATP targets were correlated to functional observations. CONCLUSION Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular KATP channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts. PMID:21586291

  11. Overexpression of NaV 1.6 channels is associated with the invasion capacity of human cervical cancer.

    Science.gov (United States)

    Hernandez-Plata, Everardo; Ortiz, Cindy S; Marquina-Castillo, Brenda; Medina-Martinez, Ingrid; Alfaro, Ana; Berumen, Jaime; Rivera, Manuel; Gomora, Juan C

    2012-05-01

    Functional activity of voltage-gated sodium channels (VGSC) has been associated to the invasion and metastasis behaviors of prostate, breast and some other types of cancer. We previously reported the functional expression of VGSC in primary cultures and biopsies derived from cervical cancer (CaC). Here, we investigate the relative expression levels of VGSC subunits and its possible role in CaC. Quantitative real-time PCR revealed that mRNA levels of Na(V) 1.6 α-subunit in CaC samples were ∼40-fold higher than in noncancerous cervical (NCC) biopsies. A Na(V) 1.7 α-subunit variant also showed increased mRNA levels in CaC (∼20-fold). All four Na(V) β subunits were also detected in CaC samples, being Na(V) β1 the most abundant. Proteins of Na(V) 1.6 and Na(V) 1.7 α-subunits were immunolocalized in both NCC and CaC biopsies and in CaC primary cultures as well; however, although in NCC sections proteins were mainly relegated to the plasma membrane, in CaC biopsies and primary cultures the respective signal was stronger and widely distributed in both cytoplasm and plasma membrane. Functional activity of Na(V) 1.6 channels in the plasma membrane of CaC cells was confirmed by whole-cell patch-clamp experiments using Cn2, a Na(V) 1.6-specific toxin, which blocked ∼30% of the total sodium current. Blocking of sodium channels VGSC with tetrodotoxin and Cn2 did not affect proliferation neither migration, but reduced by ∼20% the invasiveness of CaC primary culture cells in vitro assays. We conclude that Na(V) 1.6 is upregulated in CaC and could serve as a novel molecular marker for the metastatic behavior of this carcinoma.

  12. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart.

    Science.gov (United States)

    Marionneau, Céline; Couette, Brigitte; Liu, Jie; Li, Huiyu; Mangoni, Matteo E; Nargeot, Joël; Lei, Ming; Escande, Denis; Demolombe, Sophie

    2005-01-01

    Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (C(t)) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Nav beta 1, Nav beta 3, Cav1.3, Cav3.1 and Cav alpha 2 delta 2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cav beta 2 and Cav gamma 4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvbeta1, MinK and Cav gamma 7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.

  13. Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity

    OpenAIRE

    Bury, M.; Girault, Alban; Mégalizzi, V.; Spiegl-Kreinecker, S.; Mathieu, V.; Berger, W; Evidente, A.; Kornienko, A.; Gailly, P.; Vandier, Christophe; Kiss, R.

    2013-01-01

    International audience; Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found t...

  14. Berberine reduces cAMP-induced chloride secretion in T84 human colonic carcinoma cells through inhibition of basolateral KCNQ1 channels

    Directory of Open Access Journals (Sweden)

    Rodrigo eAlzamora

    2011-06-01

    Full Text Available Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl- secretion in distal colon. The aims of this study were to determine the molecular signalling mechanisms of action of berberine on Cl- secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80  8 M. In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl- conductance or basolateral Na+-K+-ATPase activity. Berberine stimulated p38 MAPK, PKC and PKA, but had no effect on p42/p44 MAPK and PKC. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl- secretion was partially blocked by HBDDE (65 %, an inhibitor of PKC and to a smaller extent by inhibition of p38 MAPK with SB202190 (15 %. Berberine treatment induced an increase in association between PKC and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl- secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKC-dependent pathway.

  15. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2011-01-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42\\/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCα-dependent pathway.

  16. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2012-02-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 +\\/- 8 muM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCalpha and PKA, but had no effect on p42\\/p44 MAPK and PKCdelta. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE ( approximately 65%), an inhibitor of PKCalpha and to a smaller extent by inhibition of p38 MAPK with SB202190 ( approximately 15%). Berberine treatment induced an increase in association between PKCalpha and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCalpha-dependent pathway.

  17. A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-à-go-go related gene potassium channel.

    Science.gov (United States)

    Schmalhofer, William A; Swensen, Andrew M; Thomas, Brande S; Felix, John P; Haedo, Rodolfo J; Solly, Kelli; Kiss, Laszlo; Kaczorowski, Gregory J; Garcia, Maria L

    2010-12-01

    The voltage-gated potassium channel, human Ether-à-go-go related gene (hERG), represents the molecular component of IKr, one of the potassium currents involved in cardiac action potential repolarization. Inhibition of IKr increases the duration of the ventricular action potential, reflected as a prolongation of the QT interval in the electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. Because hERG is an appropriate surrogate for IKr, hERG assays that can identify potential safety liabilities of compounds during lead identification and optimization have been implemented. Although the gold standard for hERG evaluation is electrophysiology, this technique, even with the medium capacity, automated instruments that are currently available, does not meet the throughput demands for supporting typical medicinal chemistry efforts in the pharmaceutical environment. Assays that could provide reliable molecular pharmacology data, while operating in high capacity mode, are therefore desirable. In the present study, we describe a high-capacity, 384- and 1,536-well plate, functional thallium flux assay for the hERG channel that fulfills these criteria. This assay was optimized and validated using different structural classes of hERG inhibitors. An excellent correlation was found between the potency of these agents in the thallium flux assay and in electrophysiological recordings of channel activity using the QPatch automated patch platform. Extension of this study to include 991 medicinal chemistry compounds from different internal drug development programs indicated that the thallium flux assay was a good predictor of in vitro hERG activity. These data suggest that the hERG thallium flux assay can play an important role in supporting drug development efforts.

  18. Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis)

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, S.E.; Van Bakel, I.; Craig, I.W. [Univ. of Oxford (United Kingdom)] [and others

    1995-10-10

    Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3{prime} part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus. 31 refs., 5 figs.

  19. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

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    Cinzia Ambrosi

    Full Text Available Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26 that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P. Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels

  20. Identification of a single cytosine base insertion mutation at Arg-597 of the beta subunit of the human epithelial sodium channel in a family with Liddle's disease.

    Science.gov (United States)

    Inoue, T; Okauchi, Y; Matsuzaki, Y; Kuwajima, K; Kondo, H; Horiuchi, N; Nakao, K; Iwata, M; Yokogoshi, Y; Shintani, Y; Bando, H; Saito, S

    1998-06-01

    We describe a family with Liddle's disease caused by a novel mutation of the beta subunit of the human epithelial sodium channel (ENaC). A 15-year-old Japanese female was referred to our outclinic because of hypertension. The physical examination showed no abnormal findings except mild hypertension, but the laboratory data revealed low levels of plasma renin activity, plasma aldosterone and serum potassium. A comprehensive analysis of steroid hormones showed only high levels of urinary free cortisol and 17-hydroxycorticosteroids. During loading tests, blood pressure and serum potassium responded well to triamterene and slightly to spironolactone, but did not respond to dexamethasone. In addition, the normal ratio of tetrahydrocortisol plus 5alpha-tetrahydrocortisol to tetrahydrocortisone in a 24 h urinary excretion test strongly suggested a diagnosis of Liddle's disease rather than apparent mineralocorticoid excess syndrome. DNA sequence analysis of members of this family revealed a single cytosine base insertion at Arg-597 of the beta human ENaC in the proband and her mother, leading to a loss of the last 34 amino acids from the normally encoded protein as the result of a frameshift. We conclude that a de novo cytosine insertion into the final exon of the C-terminus of the beta human ENaC is responsible for Liddle's disease in this Japanese family.

  1. Optical isomers of dihydropyridine calcium channel blockers display enantiospecific effects on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450.

    Science.gov (United States)

    Štěpánková, Martina; Krasulová, Kristýna; Dořičáková, Aneta; Kurka, Ondřej; Anzenbacher, Pavel; Dvořák, Zdeněk

    2016-11-16

    Dihydropyridine calcium channel blockers (CCBs) are used as anti-hypertensives and in the treatment of angina pectoris. Structurally, CCBs have at least one chiral center in the molecule, thereby existing in two or more different enantiomers. In the current paper we examined effects of benidipine, felodipine and isradipine enantiomers on the expression and enzyme activities of human xenobiotics-metabolizing cytochromes P450. All CCBs dose-dependently activated aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), as revealed by gene reporter assays. Activation of AhR, but not PXR, was enantiospecific. Consistently, CCBs induced CYP1A1 and CYP1A2 mRNAs, but not protein, in human hepatocytes and HepG2 cells, with following pattern: benidipine (-)>(+), isradipine (-)>(+) and felodipine (+)>(-). All CCBs induced CYP2A6, CYP2B6 and CYP3A4 mRNA and protein in human hepatocytes, and there were not differences between the enantiomers. All CCBs transformed AhR in its DNA-binding form, as revealed by electromobility shift assay. Tested CCBs inhibited enzyme activities of CYP3A4 (benidipine (+)>(-); felodipine (-)>(+); isradipine (-)-(+)) and CYP2C9 (benidipine (-)>(+); felodipine (+)>(-); isradipine (-)>(+)). The data presented here might be of toxicological and clinical importance.

  2. Variants in the human potassium channel gene (KCNN3) are associated with migraine in a high risk genetic isolate.

    Science.gov (United States)

    Cox, Hannah C; Lea, Rod A; Bellis, Claire; Carless, Melanie; Dyer, Tom; Blangero, John; Griffiths, Lyn R

    2011-12-01

    The calcium-activated potassium ion channel gene (KCNN3) is located in the vicinity of the familial hemiplegic migraine type 2 locus on chromosome 1q21.3. This gene is expressed in the central nervous system and plays a role in neural excitability. Previous association studies have provided some, although not conclusive, evidence for involvement of this gene in migraine susceptibility. To elucidate KCNN3 involvement in migraine, we performed gene-wide SNP genotyping in a high-risk genetic isolate from Norfolk Island, a population descended from a small number of eighteenth century Isle of Man 'Bounty Mutineer' and Tahitian founders. Phenotype information was available for 377 individuals who are related through the single, well-defined Norfolk pedigree (96 were affected: 64 MA, 32 MO). A total of 85 SNPs spanning the KCNN3 gene were genotyped in a sub-sample of 285 related individuals (76 affected), all core members of the extensive Norfolk Island 'Bounty Mutineer' genealogy. All genotyping was performed using the Illumina BeadArray platform. The analysis was performed using the statistical program SOLAR v4.0.6 assuming an additive model of allelic effect adjusted for the effects of age and sex. Haplotype analysis was undertaken using the program HAPLOVIEW v4.0. A total of four intronic SNPs in the KCNN3 gene displayed significant association (P migraine. Two SNPs, rs73532286 and rs6426929, separated by approximately 0.1 kb, displayed complete LD (r (2) = 1.00, D' = 1.00, D' 95% CI = 0.96-1.00). In all cases, the minor allele led to a decrease in migraine risk (beta coefficient = 0.286-0.315), suggesting that common gene variants confer an increased risk of migraine in the Norfolk pedigree. This effect may be explained by founder effect in this genetic isolate. This study provides evidence for association of variants in the KCNN3 ion channel gene with migraine susceptibility in the Norfolk genetic isolate with the rarer allelic variants conferring a

  3. Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

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    Wang, Yifan [The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071 (China); Li, Shu Jie, E-mail: shujieli@nankai.edu.cn [The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071 (China); Pan, Juncheng [The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071 (China); Che, Yongzhe, E-mail: cheli@nankai.edu.cn [The Key Laboratory of Bioactive Materials, Ministry of Education, School of Medicine, Nankai University, Tianjin 300071 (China); Yin, Jian [Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060 (China); Zhao, Qing [The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071 (China)

    2011-08-26

    Highlights: {yields} Hv1 is specifically expressed in highly metastatic human breast tumor tissues. {yields} Hv1 regulates breast cancer cytosolic pH. {yields} Hv1 acidifies extracellular milieu. {yields} Hv1 exacerbates the migratory ability of metastatic cells. -- Abstract: The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.

  4. Tumor necrosis factor-alpha (TNF-α enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes

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    Ma Fei

    2009-08-01

    Full Text Available Abstract Background We have shown functional expression of several TRP channels on human synovial cells, proposing significance in known calcium dependent proliferative and secretory responses in joint inflammation. The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha (TNF-α, EC50 1.3221 × 10-10g/L. Results Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1 TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells, (2 moderate and noxious temperature change, and (3 osmotic stress TRPV4 activation (11.5% of cells. TNF-alpha pre-treatment (1 ng/ml, 8 – 16 hr significantly increases (doubles capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i responses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca2+]i increase (temperature controlled chamber. Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase. TNF-alpha increases TRPV1 (8 hr peak and TRPV4 (12 hr peak immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions. Conclusion TNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.

  5. Downregulation of the Ca(2+)-activated K(+) channel KC a3.1 by histone deacetylase inhibition in human breast cancer cells.

    Science.gov (United States)

    Ohya, Susumu; Kanatsuka, Saki; Hatano, Noriyuki; Kito, Hiroaki; Matsui, Azusa; Fujimoto, Mayu; Matsuba, Sayo; Niwa, Satomi; Zhan, Peng; Suzuki, Takayoshi; Muraki, Katsuhiko

    2016-04-01

    The intermediate-conductance Ca(2+)-activated K(+) channel KC a3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target and biomarker for cancer. Histone deacetylase inhibitors (HDACis) have considerable potential for cancer therapy, however, the effects of HDACis on ion channel expression have not yet been investigated in detail. The results of this study showed a significant decrease in KC a3.1 transcription by HDAC inhibition in the human breast cancer cell line YMB-1, which functionally expresses KCa3.1. A treatment with the clinically available, class I, II, and IV HDAC inhibitor, vorinostat significantly downregulated KC a3.1 transcription in a concentration-dependent manner, and the plasmalemmal expression of the KC a3.1 protein and its functional activity were correspondingly decreased. Pharmacological and siRNA-based HDAC inhibition both revealed the involvement of HDAC2 and HDAC3 in KC a3.1 transcription through the same mechanism. The downregulation of KC a3.1 in YMB-1 was not due to the upregulation of the repressor element-1 silencing transcription factor, REST and the insulin-like growth factor-binding protein 5, IGFBP5. The significant decrease in KC a3.1 transcription by HDAC inhibition was also observed in the KC a3.1-expressing human prostate cancer cell line, PC-3. These results suggest that vorinostat and the selective HDACis for HDAC2 and/or HDAC3 are effective drug candidates for KC a3.1-overexpressing cancers.

  6. Three Peptide Modulators of the Human Voltage-Gated Sodium Channel 1.7, an Important Analgesic Target, from the Venom of an Australian Tarantula

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    Chun Yuen Chow

    2015-06-01

    Full Text Available Voltage-gated sodium (NaV channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7. Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM, Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.

  7. Effects on humans elicited by inhaling the fragrance of essential oils: sensory test, multi-channel thermometric study and forehead surface potential wave measurement on basil and peppermint.

    Science.gov (United States)

    Satoh, Tomoko; Sugawara, Yoshiaki

    2003-01-01

    The effects on humans inhaling the fragrance of essential oils were examined in terms of a sensory test, a multi-channel skin thermometer study and a portable forehead surface electroencephalographic (IBVA-EEG) measurement. The essential oils examined in this study were those of basil and peppermint, because our previous sensory test had indicated an opposite effect of these essential oils when mental work was undertaken; the inhalation of basil produced a more favorable impression after work than before work, whereas peppermint produced an unfavorable impression under these circumstances. For subjects administered basil or peppermint before and after mental work using an inhalator, a series of multi-channel skin thermometer studies and IBVA-EEG measurements were conducted. Using such paired odorants, our results showed that when compared between before and after mental work assigned to subjects: (1) the inhalation of basil, in which a favorable impression was predominant on the whole in terms of the sensory evaluation spectrum, was shown to be associated upward tendency in finger-tip skin temperature; (2) whereas these situations were opposite in the case of peppermint, in which the reversed (unfavorable) feature in sensory profiling was accompanied by a decrease in the magnitude of beta waves and a decrease in the finger-tip skin temperature both based on Welch's method, even at p < 0.01, implying a decreasing propensity of the aroused state and of the arousal response. The elucidation of such sensory and physiological endpoints of paired odorants would be of primary importance for human chemoreception science, because these are only rarely recorded during the same experiments, and this paradigm is highly informative about non-verbal responses to odorants.

  8. Mechanosensitive Channels

    Science.gov (United States)

    Martinac, Boris

    Living cells are exposed to a variety of mechanical stimuli acting throughout the biosphere. The range of the stimuli extends from thermal molecular agitation to potentially destructive cell swelling caused by osmotic pressure gradients. Cellular membranes present a major target for these stimuli. To detect mechanical forces acting upon them cell membranes are equipped with mechanosensitive (MS) ion channels. Functioning as molecular mechanoelectrical transducers of mechanical forces into electrical and/or chemical intracellular signals these channels play a critical role in the physiology of mechanotransduction. Studies of prokaryotic MS channels and recent work on MS channels of eukaryotes have significantly increased our understanding of their gating mechanism, physiological functions, and evolutionary origins as well as their role in the pathology of disease.

  9. Potassium Channel Ether à go-go1 Is Aberrantly Expressed in Human Liposarcoma and Promotes Tumorigenesis

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    Jin Wu

    2014-01-01

    Full Text Available The ether à go-go1 (Eag1 channel is overexpressed in a variety of cancers. However, the expression and function of Eag1 in liposarcoma are poorly understood. In the present study, the mRNA expression of Eag1 in different adipose tissue samples was examined by real-time PCR. Then, the protein expression of Eag1 in 131 different adipose tissues from 109 patients was detected by immunohistochemistry. Next, the associations between Eag1 expression and clinicopathological features of liposarcoma were analyzed. In addition, the effects of Eag1 on liposarcoma cell proliferation and cycle were evaluated by CCK-8, colony formation, xenograft mouse model, and flow cytometry, respectively. Finally, the activation of p38 mitogen-activated protein kinase (MAPK was detected by Western blot analysis to explain the detailed mechanisms of oncogenic potential of Eag1 in liposarcoma. It was found that Eag1 was aberrantly expressed in over 67% liposarcomas, with a higher frequency than in lipoma, hyperplasia, inflammation, and normal adipose tissues. However, Eag1 expression was not correlated with clinicopathological features of liposarcoma. Eag1 inhibitor imipramine or Eag1-shRNA significantly suppressed the proliferation of liposarcoma cells in vitro and in vivo, accompanying with accumulation of cells in the G1 phase. These results suggest that Eag1 plays an important role in regulating the proliferation and cell cycle of liposarcoma cells and might be a potential therapeutic target for liposarcoma.

  10. L-Carnitine Reduces in Human Conjunctival Epithelial Cells Hypertonic-Induced Shrinkage through Interacting with TRPV1 Channels

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    Noushafarin Khajavi

    2014-08-01

    Full Text Available Background/Aims: Ocular surface health depends on conjunctival epithelial (HCjE layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1 channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. Methods: Using siRNA gene silencing, Ca2+ imaging, planar patch-clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. Results: TRPV1 activation by capsaicin (CAP and an increase in osmolarity to ≈ 450 mOsM both induced increases in Ca2+ levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ fully reversed this response. Similarly, L-carnitine (1 mM also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated. Conclusion: The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na+-dependent L-carnitine transporter.

  11. Human voltage-gated proton channel hv1: a new potential biomarker for diagnosis and prognosis of colorectal cancer.

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    Yifan Wang

    Full Text Available Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s. The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA and Zn(2+ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.

  12. Improving the In Silico Assessment of Proarrhythmia Risk by Combining hERG (Human Ether-à-go-go-Related Gene) Channel-Drug Binding Kinetics and Multichannel Pharmacology.

    Science.gov (United States)

    Li, Zhihua; Dutta, Sara; Sheng, Jiansong; Tran, Phu N; Wu, Wendy; Chang, Kelly; Mdluli, Thembi; Strauss, David G; Colatsky, Thomas

    2017-02-01

    The current proarrhythmia safety testing paradigm, although highly efficient in preventing new torsadogenic drugs from entering the market, has important limitations that can restrict the development and use of valuable new therapeutics. The CiPA (Comprehensive in vitro Proarrhythmia Assay) proposes to overcome these limitations by evaluating drug effects on multiple cardiac ion channels in vitro and using these data in a predictive in silico model of the adult human ventricular myocyte. A set of drugs with known clinical torsade de pointes risk was selected to develop and calibrate the in silico model. Manual patch-clamp data assessing drug effects on expressed cardiac ion channels were integrated into the O'Hara-Rudy myocyte model modified to include dynamic drug-hERG channel (human Ether-à-go-go-Related Gene) interactions. Together with multichannel pharmacology data, this model predicts that compounds with high torsadogenic risk are more likely to be trapped within the hERG channel and show stronger reverse use dependency of action potential prolongation. Furthermore, drug-induced changes in the amount of electronic charge carried by the late sodium and L-type calcium currents was evaluated as a potential metric for assigning torsadogenic risk. Modeling dynamic drug-hERG channel interactions and multi-ion channel pharmacology improves the prediction of torsadogenic risk. With further development, these methods have the potential to improve the regulatory assessment of drug safety models under the CiPA paradigm. © 2017 American Heart Association, Inc.

  13. Calcium channels and migraine.

    Science.gov (United States)

    Pietrobon, Daniela

    2013-07-01

    Missense mutations in CACNA1A, the gene that encodes the pore-forming α1 subunit of human voltage-gated Ca(V)2.1 (P/Q-type) calcium channels, cause a rare form of migraine with aura (familial hemiplegic migraine type 1: FHM1). Migraine is a common disabling brain disorder whose key manifestations are recurrent attacks of unilateral headache that may be preceded by transient neurological aura symptoms. This review, first, briefly summarizes current understanding of the pathophysiological mechanisms that are believed to underlie migraine headache, migraine aura and the onset of a migraine attack, and briefly describes the localization and function of neuronal Ca(V)2.1 channels in the brain regions that have been implicated in migraine pathogenesis. Then, the review describes and discusses i) the functional consequences of FHM1 mutations on the biophysical properties of recombinant human Ca(V)2.1 channels and native Ca(V)2.1 channels in neurons of knockin mouse models carrying the mild R192Q or severe S218L mutations in the orthologous gene, and ii) the functional consequences of these mutations on neurophysiological processes in the cerebral cortex and trigeminovascular system thought to be involved in the pathophysiology of migraine, and the insights into migraine mechanisms obtained from the functional analysis of these processes in FHM1 knockin mice. This article is part of a Special Issue entitled: Calcium channels. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Flow injection spectrophotometric analysis of human salivary α-amylase activity using an enzyme degradation of starch-iodine complexes in flow channel and its application to human stress testing.

    Science.gov (United States)

    Ohtomo, Takao; Igarashi, Shukuro; Takagai, Yoshitaka

    2013-01-01

    Flow injection spectrophotometric analysis (FIA) of human salivary α-amylase was developed using an enzyme degradation reaction of starch-iodine complexes. In this proposed method, the salivary α-amylase, known as a human stress indicator, is directly and rapidly determined without any pretreatment. In this study, the optimum starch-iodine complexes (i.e., optimum molecular weight and amylase-amylopectin compounding ratio) were selected, and their rapid degradation in the flow channel was investigated to determine salivary amylase in the FIA system. The determination range of α-amylase was obtained from 0.25 to 5.0 kilo Novo unit per milliliter (KNU/mL), and these concentrations were equivalent to the real concentration of amylase in human saliva. The quantitative values obtained by this method were found to be highly reproducible with 1.6% (n=25) of the relative standard deviation for 1.0 KNU/mL. The detection limit (3σ) was 60 NU/mL. In addition, the method requires small volume of a sample (20 µL), and 30 samples was sequentially measured within one hour. Real human saliva collected before and after exercise was utilized to demonstrate the feasibility of human stress test and analytical performance of this approach.

  15. Expression of the calcium-activated potassium channel in upper and lower segment human myometrium during pregnancy and parturition

    Science.gov (United States)

    Gao, Lu; Cong, Binghai; Zhang, Lanmei; Ni, Xin

    2009-01-01

    Background Large conductance calcium-activated potassium channel (BKCa) plays an important role in the control of uterine contractility during pregnancy. The change from uterine quiescence to enhanced contractile activity may be associated with the spatial and temporal expression of BKCa within myometrium. The objectives of this study were to examine the expression of BKCa alpha- and beta-subunit in upper segment (US) and lower segment (LS) regions of uterus, and to investigate for the possibly differential expression of these proteins in US and LS myometrium obtained from three functional states: (1) non-pregnant (NP); (2) term pregnant not in labour (TNL) and (3) term pregnant in labour (TL). Methods Myometrial biopsies were collected from non-pregnant women at hysterectomy and pregnant women at either elective caesarean section or emergency caesarean section. Protein expression level and cellular localization of BKCa alpha- and beta-subunit in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively. Results BKCa alpha- and beta-subunit were predominantly localized to myometrial smooth muscle in both US and LS myometrium obtained from non-pregnant and pregnant patients. The level of BKCa alpha-subunit in US but not in LS was significantly higher in NP myometrium than those measured in myometrium obtained during pregnancy. Lower expression of BKCa alpha-subunit in both US and LS was found in TL than in TNL biopsies. Expression of beta-subunit in both US and LS myometrium was significantly reduced in TL group compared with those measured in TNL group. There was no significant difference in BKCa beta-subunit expression in either US or LS between NP and TNL group. Conclusion Our results suggest that expression of BKCa alpha- and beta-subunit in pregnant myometrium is reduced during labour, which is consistent with the myometrial activity at the onset of parturition. PMID:19344525

  16. Expression of the calcium-activated potassium channel in upper and lower segment human myometrium during pregnancy and parturition

    Directory of Open Access Journals (Sweden)

    Zhang Lanmei

    2009-04-01

    Full Text Available Abstract Background Large conductance calcium-activated potassium channel (BKCa plays an important role in the control of uterine contractility during pregnancy. The change from uterine quiescence to enhanced contractile activity may be associated with the spatial and temporal expression of BKCa within myometrium. The objectives of this study were to examine the expression of BKCa alpha- and beta-subunit in upper segment (US and lower segment (LS regions of uterus, and to investigate for the possibly differential expression of these proteins in US and LS myometrium obtained from three functional states: (1 non-pregnant (NP; (2 term pregnant not in labour (TNL and (3 term pregnant in labour (TL. Methods Myometrial biopsies were collected from non-pregnant women at hysterectomy and pregnant women at either elective caesarean section or emergency caesarean section. Protein expression level and cellular localization of BKCa alpha- and beta-subunit in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively. Results BKCa alpha- and beta-subunit were predominantly localized to myometrial smooth muscle in both US and LS myometrium obtained from non-pregnant and pregnant patients. The level of BKCa alpha-subunit in US but not in LS was significantly higher in NP myometrium than those measured in myometrium obtained during pregnancy. Lower expression of BKCa alpha-subunit in both US and LS was found in TL than in TNL biopsies. Expression of beta-subunit in both US and LS myometrium was significantly reduced in TL group compared with those measured in TNL group. There was no significant difference in BKCa beta-subunit expression in either US or LS between NP and TNL group. Conclusion Our results suggest that expression of BKCa alpha- and beta-subunit in pregnant myometrium is reduced during labour, which is consistent with the myometrial activity at the onset of parturition.

  17. Genetic Control of Potassium Channels.

    Science.gov (United States)

    Amin, Ahmad S; Wilde, Arthur A M

    2016-06-01

    Approximately 80 genes in the human genome code for pore-forming subunits of potassium (K(+)) channels. Rare variants (mutations) in K(+) channel-encoding genes may cause heritable arrhythmia syndromes. Not all rare variants in K(+) channel-encoding genes are necessarily disease-causing mutations. Common variants in K(+) channel-encoding genes are increasingly recognized as modifiers of phenotype in heritable arrhythmia syndromes and in the general population. Although difficult, distinguishing pathogenic variants from benign variants is of utmost importance to avoid false designations of genetic variants as disease-causing mutations.

  18. The S4-S5 linker directly couples voltage sensor movement to the activation gate in the human ether-a'-go-go-related gene (hERG) K+ channel.

    Science.gov (United States)

    Ferrer, Tania; Rupp, Jason; Piper, David R; Tristani-Firouzi, Martin

    2006-05-05

    A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.

  19. Accurate and representative decoding of the neural drive to muscles in humans with multi-channel intramuscular thin-film electrodes.

    Science.gov (United States)

    Muceli, Silvia; Poppendieck, Wigand; Negro, Francesco; Yoshida, Ken; Hoffmann, Klaus P; Butler, Jane E; Gandevia, Simon C; Farina, Dario

    2015-09-01

    Intramuscular electrodes developed over the past 80 years can record the concurrent activity of only a few motor units active during a muscle contraction. We designed, produced and tested a novel multi-channel intramuscular wire electrode that allows in vivo concurrent recordings of a substantially greater number of motor units than with conventional methods. The electrode has been extensively tested in deep and superficial human muscles. The performed tests indicate the applicability of the proposed technology in a variety of conditions. The electrode represents an important novel technology that opens new avenues in the study of the neural control of muscles in humans. We describe the design, fabrication and testing of a novel multi-channel thin-film electrode for detection of the output of motoneurones in vivo and in humans, through muscle signals. The structure includes a linear array of 16 detection sites that can sample intramuscular electromyographic activity from the entire muscle cross-section. The structure was tested in two superficial muscles (the abductor digiti minimi (ADM) and the tibialis anterior (TA)) and a deep muscle (the genioglossus (GG)) during contractions at various forces. Moreover, surface electromyogram (EMG) signals were concurrently detected from the TA muscle with a grid of 64 electrodes. Surface and intramuscular signals were decomposed into the constituent motor unit (MU) action potential trains. With the intramuscular electrode, up to 31 MUs were identified from the ADM muscle during an isometric contraction at 15% of the maximal force (MVC) and 50 MUs were identified for a 30% MVC contraction of TA. The new electrode detects different sources from a surface EMG system, as only one MU spike train was found to be common in the decomposition of the intramuscular and surface signals acquired from the TA. The system also allowed access to the GG muscle, which cannot be analysed with surface EMG, with successful identification of MU

  20. Pattern of Functional TTX-Resistant Sodium Channels Reveals a Developmental Stage of Human iPSC- and ESC-Derived Nociceptors

    Directory of Open Access Journals (Sweden)

    Esther Eberhardt

    2015-09-01

    Full Text Available Human pluripotent stem cells (hPSCs offer the opportunity to generate neuronal cells, including nociceptors. Using a chemical-based approach, we generated nociceptive sensory neurons from HUES6 embryonic stem cells and retrovirally reprogrammed induced hPSCs derived from fibroblasts. The nociceptive neurons expressed respective markers and showed tetrodotoxin-sensitive (TTXs and -resistant (TTXr voltage-gated sodium currents in patch-clamp experiments. In contrast to their counterparts from rodent dorsal root ganglia, TTXr currents of hPSC-derived nociceptors unexpectedly displayed a significantly more hyperpolarized voltage dependence of activation and fast inactivation. This apparent discrepancy is most likely due to a substantial expression of the developmentally important sodium channel NAV1.5. In view of the obstacles to recapitulate neuropathic pain in animal models, our data advance hPSC-derived nociceptors as a better model to study developmental and pathogenetic processes in human nociceptive neurons and to develop more specific small molecules to attenuate pain.

  1. Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating.

    Science.gov (United States)

    Stephan, Gabriele; Kowalski-Jahn, Maria; Zens, Christopher; Schmalzing, Günther; Illes, Peter; Hausmann, Ralf

    2016-06-01

    P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

  2. Skeletal Muscle Na+ Channel Disorders

    Directory of Open Access Journals (Sweden)

    Dina eSimkin

    2011-10-01

    Full Text Available Five inherited human disorders affecting skeletal muscle contraction have been traced to mutations in the gene encoding the voltage-gated sodium channel Nav1.4. The main symptoms of these disorders are myotonia or periodic paralysis caused by changes in skeletal muscle fiber excitability. Symptoms of these disorders vary from mild or latent disease to incapacitating or even death in severe cases. As new human sodium channel mutations corresponding to disease states become discovered, the importance of understanding the role of the sodium channel in skeletal muscle function and disease state grows.

  3. The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

    Directory of Open Access Journals (Sweden)

    Isoldi M.C.

    2004-01-01

    Full Text Available 8-Methoxy psoralen (8-MOP exerts a short-term (24 h mitogenic action, and a long-term (48-72 h anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM. The intracellular Ca2+ chelator BAPTA/AM (1 µM blocked both early (mitogenic and late (anti-proliferative and melanogenic 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

  4. APE1/Ref-1 promotes the effect of angiotensin II on Ca2+ -activated K+ channel in human endothelial cells via suppression of NADPH oxidase.

    Science.gov (United States)

    Park, Won Sun; Ko, Eun A; Jung, In Duk; Son, Youn Kyoung; Kim, Hyoung Kyu; Kim, Nari; Park, So Youn; Hong, Ki Whan; Park, Yeong-Min; Choi, Tae-Hoon; Han, Jin

    2008-10-01

    The effects of angiotensin II (Ang II) on whole-cell large conductance Ca(2+)-activated K(+) (BK(Ca)) currents was investigated in control and Apurinic/apyrimidinic endonuclease1/redox factor 1 (APE1/Ref-1)-overexpressing human umbilical vein endothelial cells (HUVECs). Ang II blocked the BK(Ca) current in a dose-dependent fashion, and this inhibition was greater in APE1/Ref-1-overexpressing HUVECs than in control HUVECs (half-inhibition values of 102.81+/-9.54 nM and 11.34+/-0.39 nM in control and APE1/Ref-1-overexpressing HUVECs, respectively). Pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or knock down of NADPH oxidase (p22 phox) using siRNA increased the inhibitory effect of Ang II on the BK(Ca) currents, similar to the effect of APE1/Ref-1 overexpression. In addition, application of Ang II increased the superoxide and hydrogen peroxide levels in the control HUVECs but not in APE1/Ref-1-overexpressing HUVECs. Furthermore, direct application of hydrogen peroxide increased BK(Ca) channel activity. Finally, the inhibitory effect of Ang II on the BK(Ca) current was blocked by an antagonist of the Ang II type 1 (AT(1)) receptor in both control and APE1/Ref-1-overexpressing HUVECs. From these results, we conclude that the inhibitory effect of Ang II on BK(Ca) channel function is NADPH oxidase-dependent and may be promoted by APE1/Ref-1.

  5. The role of potassium channels in the nitric oxide-induced relaxation of human airway smooth muscle of passively sensitization by serum from allergic asthmatic patients

    Institute of Scientific and Technical Information of China (English)

    Tao Ye; Yongjian Xu; Zhenxiang Zhang; Xiansheng Liu; Zhao Yang; Baoan Gao

    2006-01-01

    Objective: To investigate the role of large Ca2+-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide (NO) in normal and passively sensitized human airway smooth muscle (HASM) with serum from asthmatic patients. Methods: The effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique. Results: Showed that (1)In the control group and passively sensitized group, Kv blocker (4-AP) cause concentration-dependent augmentation in the contraction induced by histamine (1 ×10-4 mol/L), (P < 0.05), but Glib (1 × 10-2 mol/L)and TEA (1×10-3 mol/L) have no significant effects on the contraction induced by histamine (1×10-4 mol/L). The maximum tension induced by histamine in passively sensitized group is higher than that in the control group (P < 0.05). (2) NO-donor Sodium Nitroprusside (SNP) bring about significant relaxation in normal and passively sensitized HASM rings (P < 0.05). Relaxations of passively sensitized airway rings [ (29.4 ± 3.3)% ] were significant less than those of normal HASM rings [ (44.1 ± 10.2)% ], (P <0.05).(3) Glib(1×10-2 mol/L)have no significant effect on the relaxations induced by SNP(1×10-4 mol/L). 4-AP(1×10-2 mol/L) inhibited relaxation induced by SNP (1×10-4 mol/L), (P < 0.01). TEA (1×10-3 mol/L) inhibited relaxation induced by SNP (1×10-4mol/L) (P < 0.05), and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM, (P <0.05). Conclusion: It was concluded that SNP(NO-donor) relaxed the contraction of HASM partly via BKca channel opening. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.

  6. Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells.

    NARCIS (Netherlands)

    Thebault, S.C.; Lemonnier, L.; Bidaux, G.; Flourakis, M.; Bavencoffe, A.; Gordienko, D.; Roudbaraki, M.; Delcourt, P.; Panchin, Y.; Shuba, Y.; Skryma, R.; Prevarskaya, N.

    2005-01-01

    Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the pro

  7. Capillary electrophoresis and 5-channel LIF detection of a 26plex autosomal STR assay for human identification.

    Science.gov (United States)

    Hill, Carolyn R

    2012-01-01

    Multiplex polymerase chain reaction (PCR) is a common method used for DNA typing in forensic and paternity cases. There are numerous commercial short tandem repeat (STR) multiplex assays currently available to the forensic community. These assays amplify the core Combined DNA Index System (CODIS) STR loci for entry into the US. DNA database. Additional non-CODIS loci, which are considered genetically unlinked to the CODIS loci, can be useful in resolving challenging cases such as missing persons and mass disaster victim identification, paternity testing, and immigration testing. An STR multiplex has been successfully developed with 25 non-CODIS autosomal loci plus the sex-typing locus amelogenin for a total of 26 loci in a single 26plex amplification reaction. This chapter will focus on the preparation and the use of the 26plex assay with DNA samples for the purpose of human identification.

  8. Upregulation of the transient receptor potential ankyrin 1 ion channel in the inflamed human and mouse colon and its protective roles.

    Directory of Open Access Journals (Sweden)

    József Kun

    Full Text Available Transient Receptor Potential Ankyrin 1 (TRPA1 channels are localized on sensory nerves and several non-neural cells, but data on their functional significance are contradictory. We analysed the presence and alterations of TRPA1 in comparison with TRP Vanilloid 1 (TRPV1 at mRNA and protein levels in human and mouse intact and inflamed colons. The role of TRPA1 in a colitis model was investigated using gene-deficient mice. TRPA1 and TRPV1 expressions were investigated in human colon biopsies of healthy subjects and patients with inflammatory bowel diseases (IBD: ulcerative colitis, Crohn's disease with quantitative PCR and immunohistochemistry. Mouse colitis was induced by oral 2% dextran-sulphate (DSS for 10 days. For investigating the functions of TRPA1, Disease Activity Index (weight loss, stool consistency, blood content was determined in C57BL/6-based Trpa1-deficient (knockout: KO and wildtype (WT mice. Sensory neuropeptides, their receptors, and inflammatory cytokines/chemokines were determined with qPCR or Luminex. In human and mouse colons TRPA1 and TRPV1 are located on epithelial cells, macrophages, enteric ganglia. Significant upregulation of TRPA1 mRNA was detected in inflamed samples. In Trpa1 KO mice, Disease Activity Index was significantly higher compared to WTs. It could be explained by the greater levels of substance P, neurokinins A and B, neurokinin 1 receptor, pituitary adenylate-cyclase activating polypeptide, vasoactive intestinal polypeptide, and also interleukin-1beta, macrophage chemoattractant protein-1, monokine induced by gamma interferon-1, tumor necrosis factor-alpha and B-lymphocyte chemoattractant in the distal colon. TRPA1 is upregulated in colitis and its activation exerts protective roles by decreasing the expressions of several proinflammatory neuropeptides, cytokines and chemokines.

  9. Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype

    DEFF Research Database (Denmark)

    Hougaard, Charlotte; Hammami, Sofia; Eriksen, Birgitte L;

    2012-01-01

    ]pyrimidines, act either as activators or as inhibitors of the human K(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) value of 31 nM. In contrast, their (+)-enantiomers are 40 to 100 times less active. Both (-)-CM-TPMF and (-)-B......-TPMF are subtype-selective, with 10- to 20-fold discrimination toward other K(Ca)2 channels and the K(Ca)3 channel. Coapplication experiments reveal competitive-like functional interactions between the effects of (-)-CM-TPMF and (-)-B-TPMF. Despite belonging to a different chemical class than GW542573X, the K(Ca)2......-TPMF is 10 times more potent on K(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide...

  10. Evaluation and Verification of Channel Transmission Characteristics of Human Body for Optimizing Data Transmission Rate in Electrostatic-Coupling Intra Body Communication System: A Comparative Analysis.

    Directory of Open Access Journals (Sweden)

    Yuhwai Tseng

    Full Text Available Intra-body communication is a new wireless scheme for transmitting signals through the human body. Understanding the transmission characteristics of the human body is therefore becoming increasingly important. Electrostatic-coupling intra-body communication system in a ground-free situation that integrate electronic products that are discretely located on individuals, such as mobile phones, PDAs, wearable computers, and biomedical sensors, are of particular interest.The human body is modeled as a simplified Resistor-Capacitor network. A virtual ground between the transmitter and receiver in the system is represented by a resister-capacitor network. Value of its resistance and capacitance are determined from a system perspective. The system is characterized by using a mathematical unit step function in digital baseband transmission scheme with and without Manchester code. As a result, the signal-to-noise and to-intersymbol-interference ratios are improved by manipulating the load resistor. The data transmission rate of the system is optimized. A battery-powered transmitter and receiver are developed to validate the proposal.A ground-free system fade signal energy especially for a low-frequency signal limited system transmission rate. The system transmission rate is maximized by simply manipulating the load resistor. Experimental results demonstrate that for a load resistance of 10k-50k Ω, the high-pass 3 dB frequency of the band-pass channel is 400kHz-2MHz in the worst-case scenario. The system allows a Manchester-coded baseband signal to be transmitted at speeds of up to 20M bit per second with signal-to-noise and signal-to-intersymbol-interference ratio of more than 10 dB.The human body can function as a high speed transmission medium with a data transmission rate of 20Mbps in an electrostatic-coupling intra-body communication system. Therefore, a wideband signal can be transmitted directly through the human body with a good signal

  11. Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit.

    Science.gov (United States)

    Maltsev, Victor A; Kyle, John W; Undrovinas, Albertas

    2009-05-01

    Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I(NaL)) for the function of both normal and, especially, failing myocardium, in which I(NaL) is reportedly increased. While recent molecular studies identified the cardiac Na(+) channel (NaCh) alpha subunit isoform (Na(v)1.5) as a major contributor to I (NaL), the molecular mechanisms underlying alterations of I(NaL) in heart failure (HF) are still unknown. Here we tested the hypothesis that I(NaL) is modulated by the NaCh auxiliary beta subunits. tsA201 cells were transfected simultaneously with human Na(v)1.5 (former hH1a) and cardiac beta(1) or beta(2) subunits, and whole-cell patch-clamp experiments were performed. We found that I(NaL) decay kinetics were significantly slower in cells expressing alpha + beta(1) (time constant tau = 0.73 +/- 0.16 s, n = 14, mean +/- SEM, P < 0.05) but remained unchanged in cells expressing alpha + beta(2) (tau = 0.52 +/- 0.09 s, n = 5), compared with cells expressing Na(v)1.5 alone (tau = 0.54 +/- 0.09 s, n = 20). Also, beta(1), but not beta(2), dramatically increased I(NaL) relative to the maximum peak current, I(NaT) (2.3 +/- 0.48%, n = 14 vs. 0.48 +/- 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary beta(1) subunit modulates I(NaL), produced by the human cardiac Na(+) channel Na(v)1.5 by slowing its decay and increasing I(NaL) amplitude relative to I(NaT). Because expression of Na(v)1.5 reportedly decreases but beta(1) remains unchanged in chronic HF, the relatively higher expression of beta(1) may contribute to the known I(NaL) increase in HF via the modulation mechanism found in this study.

  12. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Tada, Hiroyuki [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@umin.ac.jp [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Kanaya, Sousuke; Hamaji, Nozomu; Sato, Hisae; Shimauchi, Hidetoshi [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2010-04-16

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.

  13. Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

    Science.gov (United States)

    Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

    2017-03-01

    Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and

  14. Positron Channeling

    CERN Document Server

    Badikyan, Karen

    2016-01-01

    The possibility of channeling the low-energy relativistic positrons around separate crystallographic axes with coaxial symmetry of negative ions in some types of crystals is shown. The process of annihilation of positrons with electrons of medium was studied in detail.

  15. Brands & Channels

    Institute of Scientific and Technical Information of China (English)

    Alice Yang

    2009-01-01

    @@ "Brands" and "Channels" are the two most important things in Ku-Hai Chen's eyes when doing business with Main-land China. Ku-Hai Chen, Executive Director of the International Trade Institute of Taiwan External Trade Development Council (TAITRA), flies frequently between Chinese Taipei and Mainland China, and was in Beijing earlier this month for his seminar.

  16. The Role of Gap Junction Channels During Physiologic and Pathologic Conditions of the Human Central Nervous System

    Science.gov (United States)

    Basilio, Daniel; Sáez, Juan C.; Orellana, Juan A.; Raine, Cedric S.; Bukauskas, Feliksas; Bennett, Michael V. L.; Berman, Joan W.

    2013-01-01

    Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP3, and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system. PMID:22438035

  17. The role of gap junction channels during physiologic and pathologic conditions of the human central nervous system.

    Science.gov (United States)

    Eugenin, Eliseo A; Basilio, Daniel; Sáez, Juan C; Orellana, Juan A; Raine, Cedric S; Bukauskas, Feliksas; Bennett, Michael V L; Berman, Joan W

    2012-09-01

    Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.

  18. Improved functional expression of human cardiac kv1.5 channels and trafficking-defective mutants by low temperature treatment.

    Directory of Open Access Journals (Sweden)

    Wei-Guang Ding

    Full Text Available We herein investigated the effect of low temperature exposure on the expression, degradation, localization and activity of human Kv1.5 (hKv1.5. In hKv1.5-expressing CHO cells, the currents were significantly increased when cultured at a reduced temperature (28°C compared to those observed at 37°C. Western blot analysis indicated that the protein levels (both immature and mature proteins of hKv1.5 were significantly elevated under the hypothermic condition. Treatment with a proteasome inhibitor, MG132, significantly increased the immature, but not the mature, hKv1.5 protein at 37°C, however, there were no changes in either the immature or mature hKv1.5 proteins at low temperature following MG132 exposure. These observations suggest that the enhancement of the mature hKv1.5 protein at reduced temperature may not result from the inhibition of proteolysis. Moreover, the hKv1.5 fluorescence signal in the cells increased significantly on the cell surface at 28°C versus those cultured at 37°C. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5, which showed considerable overlap with the trans-Golgi component. Experiments using tunicamycin, an inhibitor of N-glycosylation, indicated that the N-glycosylation of hKv1.5 is more effective at 28°C than at 37°C. Finally, the hypothermic treatment also rescued the protein expression and currents of trafficking-defective hKv1.5 mutants. These results indicate that low temperature exposure stabilizes the protein in the cellular organelles or on the plasma membrane, and modulates its maturation and trafficking, thus enhancing the currents of hKv1.5 and its trafficking defect mutants.

  19. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key compon

  20. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key

  1. Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

    Science.gov (United States)

    Morera, Francisco J; Saravia, Julia; Pontigo, Juan Pablo; Vargas-Chacoff, Luis; Contreras, Gustavo F; Pupo, Amaury; Lorenzo, Yenisleidy; Castillo, Karen; Tilegenova, Cholpon; Cuello, Luis G; Gonzalez, Carlos

    2015-11-01

    Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. TRP channels and psychiatric disorders.

    Science.gov (United States)

    Chahl, Loris A

    2011-01-01

    Depression and schizophrenia are major psychiatric disorders that cause much human suffering. Current treatments have major limitations and new drug targets are eagerly sought. Study of transient receptor potential (TRP) channels in these disorders is at an early stage and the potential of agents that activate or inhibit these channels remains speculative. The findings that TRPC6 channels promote dendritic growth and are selectively activated by hyperforin, the key constitutent of St John's wort, suggest that TRPC6 channels might prove to be a new target for antidepressant drug development. There is now considerable evidence that TRPV1 antagonists have anxiolytic activity but there is no direct evidence that they have antidepressant activity. There is also no direct evidence that TRP channels play a role in schizophrenia. However, the findings that TRPC channels are involved in neuronal development and fundamental synaptic mechanisms, and that TRPV1 channels play a role in central dopaminergic and cannabinoid mechanisms is suggestive of potential roles of these channels in schizophrenia. Investigation of TRP channels in psychiatric disorders holds the promise of yielding further understanding of the aetiology of psychiatric disorders and the development of new drug treatments.

  3. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wei-Wan Zheng

    2016-01-01

    Full Text Available Recent studies suggest that the epithelial sodium channel (ENaC is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl. Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO. Moreover, nitric oxide synthase (eNOS phosphorylation (Ser 1177 levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

  4. L-type Ca(2+) channels in mood, cognition and addiction: integrating human and rodent studies with a focus on behavioural endophenotypes.

    Science.gov (United States)

    Kabir, Z D; Lee, A S; Rajadhyaksha, A M

    2016-10-15

    Brain Cav 1.2 and Cav 1.3 L-type Ca(2+) channels play key physiological roles in various neuronal processes that contribute to brain function. Genetic studies have recently identified CACNA1C as a candidate risk gene for bipolar disorder (BD), schizophrenia (SCZ), major depressive disorder (MDD) and autism spectrum disorder (ASD), and CACNA1D for BD and ASD, suggesting a contribution of Cav 1.2 and Cav 1.3 Ca(2+) signalling to the pathophysiology of neuropsychiatric disorders. Once considered sole clinical entities, it is now clear that BD, SCZ, MDD and ASD share common phenotypic features, most likely due to overlapping neurocircuitry and common molecular mechanisms. A major future challenge lies in translating the human genetic findings to pathological mechanisms that are translatable back to the patient. One approach for tackling such a daunting scientific endeavour for complex behaviour-based neuropsychiatric disorders is to examine intermediate biological phenotypes in the context of endophenotypes within distinct behavioural domains. This will better allow us to integrate findings from genes to behaviour across species, and improve the chances of translating preclinical findings to clinical practice.

  5. Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene-KCNA1 (Kv1.1)

    Energy Technology Data Exchange (ETDEWEB)

    Browne, D.L.; Gancher, S.T.; Nutt, J.G. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others

    1994-09-01

    Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. One type of EA (MIM No.160120) displays autosomal dominant inheritance and is characterized by episodes of ataxia lasting seconds to minutes with myokymia (rippling of small muscles) evident between attacks. Genetic linkage studies in 4 families suggested localization of an EA/myokymia gene near the K{sup +} channel gene KCNA1 (Kv1.1) on chromosome 12p. Chemical cleavage mismatch and DNA sequence analysis of the KCNA1 coding region in these families identified 4 different missense point mutations present in the heterozygous state. The mutations found were Val174Phe, Arg239Ser, Phe249Ile and Val408Ala; the residue numbers correspond to those in the published amino acid sequence of KCNA1 (Genbank Accession No. L02750). Each of these mutations affects an amino acid residue that is invariant among Drosophila melanogaster, mouse, rat and human, The mutations were present in the affected members of the family and absent in all of the unaffected members and in at least 70 unrelated control individuals. These data strongly suggest that EA/myokymia can result from mutations in the KCNA1 gene.

  6. Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

    Science.gov (United States)

    Khatun, Anowara; Fujimoto, Mayu; Kito, Hiroaki; Niwa, Satomi; Suzuki, Takayoshi; Ohya, Susumu

    2016-01-01

    Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells. PMID:27973439

  7. Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

    Directory of Open Access Journals (Sweden)

    Anowara Khatun

    2016-12-01

    Full Text Available Vitamin D (VD reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

  8. Gypenosides Induce Apoptosis by Ca2+ Overload Mediated by Endoplasmic-Reticulum and Store-Operated Ca2+ Channels in Human Hepatoma Cells

    Science.gov (United States)

    Sun, Da-Peng; Li, Xiao-Xi; Liu, Xin-Li; Zhao, Dan; Qiu, Feng-Qi; Li, Yan

    2013-01-01

    Abstract Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca2+-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca2+ concentration ([Ca2+]i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca2+]i level. Gyp-increased [Ca2+]i level was partly inhibited by removal of extracellular Ca2+ by Ca2+ chelator EGTA, store-operated Ca2+ channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca2+-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca2+ response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca2+-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca2+]i increase and apoptosis in HepG2 cells. Exposure to 300 μg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca2+]i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca2+ release from ER stores and increased store-operated Ca2+ entry. PMID:25310348

  9. Nonlinear channelizer

    Science.gov (United States)

    In, Visarath; Longhini, Patrick; Kho, Andy; Neff, Joseph D.; Leung, Daniel; Liu, Norman; Meadows, Brian K.; Gordon, Frank; Bulsara, Adi R.; Palacios, Antonio

    2012-12-01

    The nonlinear channelizer is an integrated circuit made up of large parallel arrays of analog nonlinear oscillators, which, collectively, serve as a broad-spectrum analyzer with the ability to receive complex signals containing multiple frequencies and instantaneously lock-on or respond to a received signal in a few oscillation cycles. The concept is based on the generation of internal oscillations in coupled nonlinear systems that do not normally oscillate in the absence of coupling. In particular, the system consists of unidirectionally coupled bistable nonlinear elements, where the frequency and other dynamical characteristics of the emergent oscillations depend on the system's internal parameters and the received signal. These properties and characteristics are being employed to develop a system capable of locking onto any arbitrary input radio frequency signal. The system is efficient by eliminating the need for high-speed, high-accuracy analog-to-digital converters, and compact by making use of nonlinear coupled systems to act as a channelizer (frequency binning and channeling), a low noise amplifier, and a frequency down-converter in a single step which, in turn, will reduce the size, weight, power, and cost of the entire communication system. This paper covers the theory, numerical simulations, and some engineering details that validate the concept at the frequency band of 1-4 GHz.

  10. Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gβγ Protein Subunits.

    Science.gov (United States)

    Berecki, Géza; Motin, Leonid; Adams, David J

    2016-01-01

    Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.

  11. Caracterización del canal epitelial de sodio en sinciciotrofoblasto de placenta humana preeclamptica Characterization of the epithelial sodium channel in human pre-eclampsia syncytiotrophoblast

    Directory of Open Access Journals (Sweden)

    Silvana del Mónaco

    2006-02-01

    epithelial Na channel (ENaC in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon, abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle´s syndrome whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of a-ENaC was cloned and sequenced having a 100% identity with the sequence of a-ENaC obtained from GenBankTM (SCNN1A, accession number Z92981. We found that the transcription of the a-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaC channels in Bewo cells. We observed currents that were blocked by 10 µM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 ± 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.

  12. Demonstration of physical proximity between the N terminus and the S4-S5 linker of the human ether-a-go-go-related gene (hERG) potassium channel.

    Science.gov (United States)

    de la Peña, Pilar; Alonso-Ron, Carlos; Machín, Angeles; Fernández-Trillo, Jorge; Carretero, Luis; Domínguez, Pedro; Barros, Francisco

    2011-05-27

    Potassium channels encoded by the human ether-à-go-go-related gene (hERG) contribute to cardiac repolarization as a result of their characteristic gating properties. The hERG channel N terminus acts as a crucial determinant in gating. It is also known that the S4-S5 linker couples the voltage-sensing machinery to the channel gate. Moreover, this linker has been repeatedly proposed as an interaction site for the distal portion of the N terminus controlling channel gating, but direct evidence for such an interaction is still lacking. In this study, we used disulfide bond formation between pairs of engineered cysteines to demonstrate the close proximity between the beginning of the N terminus and the S4-S5 linker. Currents from channels with introduced cysteines were rapidly and strongly attenuated by an oxidizing agent, this effect being maximal for cysteine pairs located around amino acids 3 and 542 of the hERG sequence. The state-dependent modification of the double-mutant channels, but not the single-cysteine mutants, and the ability to readily reverse modification with the reducing agent dithiothreitol indicate that a disulfide bond is formed under oxidizing conditions, locking the channels in a non-conducting state. We conclude that physical interactions between the N-terminal-most segment of the N terminus and the S4-S5 linker constitute an essential component of the hERG gating machinery, thus providing a molecular basis for previous data and indicating an important contribution of these cytoplasmic domains in controlling its unusual gating and hence determining its physiological role in setting the electrical behavior of cardiac and other cell types.

  13. Local anesthetic interaction with human ether-a-go-go-related gene (HERG) channels: role of aromatic amino acids Y652 and F656

    DEFF Research Database (Denmark)

    Siebrands, Cornelia C; Schmitt, Nicole; Friederich, Patrick

    2005-01-01

    was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. METHODS: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells...... by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. RESULTS: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order...... bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine...

  14. The expression of hyperpolarization activated cyclic nucleotide gated (HCN channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation

    Directory of Open Access Journals (Sweden)

    Page Carly

    2008-08-01

    Full Text Available Abstract Background Aim of this study was to test the hypothesis that levels of hyperpolarization activated cyclic nucleotide gated channels 1 to 4 (HCN1-4 are linked to the reproductive age of the ovary. Methods Young, adult, and reproductively aged ovaries were collected from Sprague-Dawley rats. RT-PCR and western blot analysis of ovaries was performed to investigate the presence of mRNA and total protein for HCN1-4. Immunohistochemistry with semiquantitative H score analysis was performed using whole ovarian histologic sections. Results RT-PCR analysis showed the presence of mRNA for HCN1-4. Western blot analysis revealed HCN1-3 proteins in all ages of ovarian tissues. Immunohistochemistry with H score analysis demonstrated distinct age-related changes in patterns of HCN1-3 in the oocytes, granulosa cells, theca cells, and corpora lutea. HCN4 was present only in the oocytes, with declining levels during the reproduction lifespan. Conclusion The evidence presented here demonstrates cell-type and developmental age patterns of HCN1-4 channel expression in rat ovaries. Based on this, we hypothesize that HCN channels have functional significance in rat ovaries and may have changing roles in reproductive aging.

  15. Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.

    Science.gov (United States)

    Rosa, Paolo; Sforna, Luigi; Carlomagno, Silvia; Mangino, Giorgio; Miscusi, Massimo; Pessia, Mauro; Franciolini, Fabio; Calogero, Antonella; Catacuzzeno, Luigi

    2017-09-01

    Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478-2488, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Differential state-dependent modification of rat Na{sub v}1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin

    Energy Technology Data Exchange (ETDEWEB)

    He, Bingjun [College of Life Sciences, Nankai University, Tianjin 300071 (China); Soderlund, David M., E-mail: dms6@cornell.edu [Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (United States)

    2011-12-15

    We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}1 and {beta}2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na{sub v}1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent 'late' currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were {approx} 25 mV for tefluthrin and {approx} 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of {approx} 5-10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na{sub v}1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by {approx} 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na{sub v}1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na{sub v}1.6 channels

  17. Activation of ATP-sensitive potassium channels facilitates the function of human endothelial colony-forming cells via Ca(2+) /Akt/eNOS pathway.

    Science.gov (United States)

    Wu, Yan; He, Meng-Yu; Ye, Jian-Kui; Ma, Shu-Ying; Huang, Wen; Wei, Yong-Yue; Kong, Hui; Wang, Hong; Zeng, Xiao-Ning; Xie, Wei-Ping

    2017-03-01

    Accumulating data, including those from our laboratory, have shown that the opening of ATP-sensitive potassium channels (KATP ) plays a protective role in pulmonary vascular diseases (PVD). As maintainers of the endothelial framework, endothelial colony-forming cells (ECFCs) are considered excellent candidates for vascular regeneration in cases of PVD. Although KATP openers (KCOs) have been demonstrated to have beneficial effects on endothelial cells, the impact of KATP on ECFC function remains unclear. Herein, this study investigated whether there is a distribution of KATP in ECFCs and what role KATP play in ECFC modulation. By human ECFCs isolated from adult peripheral blood, KATP were confirmed for the first time to express in ECFCs, comprised subunits of Kir (Kir6.1, Kir6.2) and SUR2b. KCOs such as the classical agent nicorandil (Nico) and the novel agent iptakalim (Ipt) notably improved the function of ECFCs, promoting cell proliferation, migration and angiogenesis, which were abolished by a non-selective KATP blocker glibenclamide (Gli). To determine the underlying mechanisms, we investigated the impacts of KCOs on CaMKII, Akt and endothelial nitric oxide synthase pathways. Enhanced levels were detected by western blotting, which were abrogated by Gli. This suggested an involvement of Ca(2+) signalling in the regulation of ECFCs by KATP . Our findings demonstrated for the first time that there is a distribution of KATP in ECFCs and KATP play a vital role in ECFC function. The present work highlighted a novel profile of KATP as a potential target for ECFC modulation, which may hold the key to the treatment of PVD. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  18. A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

    Science.gov (United States)

    Akimoto, Madoka; Zhang, Zaiyong; Boulton, Stephen; Selvaratnam, Rajeevan; VanSchouwen, Bryan; Gloyd, Melanie; Accili, Eric A; Lange, Oliver F; Melacini, Giuseppe

    2014-08-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

  19. The human ether-a'-go-go related gene (hERG) K+ channel blockade by the investigative selective-serotonin reuptake inhibitor CONA-437: limited dependence on S6 aromatic residues.

    Science.gov (United States)

    Alexandrou, A J; Milnes, J T; Sun, S Z; Fermini, B; Kim, S C; Jenkinson, S; Leishman, D J; Witchel, H J; Hancox, J C; Leaney, J L

    2014-08-01

    Diverse non-cardiac drugs adversely influence cardiac electrophysiology by inhibiting repolarising K(+) currents mediated by channels encoded by the human ether-a-go-go-related gene (hERG). In this study, pharmacological blockade of hERG K(+) channel current (I(hERG)) by a novel investigative serotonin-selective reuptake inhibitor (SSRI), CONA-437, was investigated. Whole-cell patch-clamp measurements of I(hERG) were made from human embryonic kidney (HEK 293) cells expressing wild-type (WT) or mutant forms of the hERG channel. With a step-ramp voltage-command, peak I(hERG) was inhibited with an IC(50) of 1.34 μM at 35 ±1°C; the IC(50) with the same protocol was not significantly different at room temperature. Voltage-command waveform selection had only a modest effect on the potency of I(hERG) block: the IC50 with a ventricular action potential command was 0.72 μM. I(hERG) blockade developed rapidly with time following membrane depolarisation and showed a weak dependence on voltage, accompanied by a shift of ≈ -5 mV in voltage-dependence of activation. There was no significant effect of CONA-437 on voltage-dependence of I(hERG) inactivation, though at some voltages an apparent acceleration of the time-course of inactivation was observed. Significantly, mutation of the S6 aromatic amino acid residues Y652 and F656 had only a modest effect on I(hERG) blockade by CONA-437 (a 3-4 fold shift in affinity). CONA-437 at up to 30 μM had no significant effect on either Nav1.5 sodium channels or L-type calcium channels. In conclusion, the novel SSRI CONA-437 is particularly notable as a gating-dependent hERG channel inhibitor for which neither S6 aromatic amino-acid constituent of the canonical drug binding site on the hERG channel appears obligatory for I(hERG) inhibition to occur.

  20. Use of navigation channels by Lake Sturgeon: Does channelization increase vulnerability of fish to ship strikes?

    Science.gov (United States)

    Bennion, David H.; Roseman, Edward F.; Holbrook, Christopher M.; Boase, James C.; Chiotti, Justin A.; Thomas, Michael V.; Wills, Todd C.; Drouin, Richard G.; Kessel, Steven T.; Krueger, Charles C.

    2017-01-01

    Channelization for navigation and flood control has altered the hydrology and bathymetry of many large rivers with unknown consequences for fish species that undergo riverine migrations. In this study, we investigated whether altered flow distributions and bathymetry associated with channelization attracted migrating Lake Sturgeon (Acipenser fulvescens) into commercial navigation channels, potentially increasing their exposure to ship strikes. To address this question, we quantified and compared Lake Sturgeon selection for navigation channels vs. alternative pathways in two multi-channel rivers differentially affected by channelization, but free of barriers to sturgeon movement. Acoustic telemetry was used to quantify Lake Sturgeon movements. Under the assumption that Lake Sturgeon navigate by following primary flow paths, acoustic-tagged Lake Sturgeon in the more-channelized lower Detroit River were expected to choose navigation channels over alternative pathways and to exhibit greater selection for navigation channels than conspecifics in the less-channelized lower St. Clair River. Consistent with these predictions, acoustic-tagged Lake Sturgeon in the more-channelized lower Detroit River selected the higher-flow and deeper navigation channels over alternative migration pathways, whereas in the less-channelized lower St. Clair River, individuals primarily used pathways alternative to navigation channels. Lake Sturgeon selection for navigation channels as migratory pathways also was significantly higher in the more-channelized lower Detroit River than in the less-channelized lower St. Clair River. We speculated that use of navigation channels over alternative pathways would increase the spatial overlap of commercial vessels and migrating Lake Sturgeon, potentially enhancing their vulnerability to ship strikes. Results of our study thus demonstrated an association between channelization and the path use of migrating Lake Sturgeon that could prove important for

  1. Post-Translational Modifications of TRP Channels

    Directory of Open Access Journals (Sweden)

    Olaf Voolstra

    2014-04-01

    Full Text Available Transient receptor potential (TRP channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

  2. Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

    Science.gov (United States)

    Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang

    2016-10-15

    In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

  3. 人体前臂组织各向异性的人体通信信道模型%Intra-body communication channel model based on anisotropic tissue in human forearm

    Institute of Scientific and Technical Information of China (English)

    刘益和; 张双; 秦雨萍; 张绍祥; 谭立文

    2014-01-01

    人体前臂是一个近似的圆柱体结构,由组织包裹组成。这些组织有的各向同性,如皮肤,脂肪;有的各向异性,如肌肉等。它们对人体电流信号的传播与分布有着极大的影响,特别是肌肉组织。应用麦克斯韦方程,结合人体组织特性和准静态条件下的边界条件,在柱坐标系下建立了基于人体组织特性的信道模型。用该模型,结合人体组织各向特性的电参数(肌肉),在MATLAB2010a 上分别计算出具有组织特性的信道模型和不具有组织特性的信道模型的结果。然后与在人体右前臂测量得到的数据相比较,发现加入组织特性的信道模型的增益曲线与实验数据保持高度一致,模型的平均误差比各向同性的信道模型误差下降了2%,最大误差也下降了3%,进一步降低了模型的失真率。%Human forearm approximates to a cylindrical structure;it is formed through encapsulating layers of human tissues. Some of them are isotropic,such as skin and fat,while the others like muscle are anisotropic.They have great effects on trans-mission and distribution of the current signal in human body,especially muscular tissue.Combining human tissues’characteris-tics and boundary conditions under the quasi-static condition,the channel model based on human tissues’characteristics is built in the cylindrical coordinate system by means of Maxwell equation.In combination with electric parameters of human anisotropic tis-sues (muscle),the obtained channel model is used to derive the channel model with human tissues’characteristics and the one without human tissues’characteristics respectively in MATLAB2010a.Afterwards,these results are compared with the data ob-tained from measurement on human right forearm.The comparison shows that the gain curve of the channel model with human tissues’characteristics is highly consistent with experimental data.Compared with the isotropic channel

  4. Investigations of the Navβ1b sodium channel subunit in human ventricle; functional characterization of the H162P Brugada Syndrome mutant

    DEFF Research Database (Denmark)

    Yuan, Lei; Koivumaki, Jussi; Liang, Bo

    2014-01-01

    Brugada Syndrome (BrS) is a rare inherited disease which can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1-4 subunits have...... to support the notion that BrS can be linked to the function of Navβ1b and is associated with loss-of-function of the cardiac sodium channel....

  5. Bi-channel Video Fusion Human Invasion Detection Based on Neural Network%基于神经网络的双通道视频融合人员入侵检测

    Institute of Scientific and Technical Information of China (English)

    王志明; 张丽; 包宏

    2012-01-01

    To overcome the low detection precision of single visible camera, a human, detection system by fusion of bi-channel video is proposed. Visible and infrared video are obtained by a visible camera and a thermal infrared video of the same scene. Motion regions are detected separately in two videos by neural network background model with adaptive learning rate. Detected results of two channels are fused by image registration and logical "or" operation and noise are removed by Gauss filter. Human like rectangular regions are detected efficiently by using integral image. Experimental results show that, bi-channel video fusion can detect pedestrian and bicycle with presion of 98%, which is more reliable than a single channel video.%为克服单个可见光摄像头检测准确率低的问题,提出一种融合双通道视频的人员检测系统.由可见光摄像头和红外热像仪分别获取同一场景的可见光和红外线视频数据,使用自适应学习速率的神经网络背景模型在2个通道中分别检测运动区域.通过图像配准对2个通道的结果进行“或”融合,并采用高斯滤波以消除噪声,利用积分图像快速检测近似长方形响应的人体区域.实验结果表明,该系统对行人和骑自行车人员的检测准确率达到98%,比单一通道具有更高的可靠性.

  6. Dendritic HCN channels shape excitatory postsynaptic potentials at the inner hair cell afferent synapse in the mammalian cochlea.

    Science.gov (United States)

    Yi, Eunyoung; Roux, Isabelle; Glowatzki, Elisabeth

    2010-05-01

    Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the auditory pathway, is specialized for rapid and reliable signaling. Here we investigated the properties of a hyperpolarization-activated current (I(h)), expressed in the afferent dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity. We used whole cell patch-clamp recordings from afferent dendrites directly where they contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials (EPSPs) of variable amplitude (1-35 mV) were found with 10-90% rise times of about 1 ms and time constants of decay of about 5 ms at room temperature. Current-voltage relations recorded in afferent dendrites revealed I(h). The pharmacological profile and reversal potential (-45 mV) indicated that I(h) is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN channel subunits HCN1, HCN2, and HCN4 were found to be expressed in afferent dendrites using immunolabeling. Raising intracellular cAMP levels sped up the activation kinetics, increased the magnitude of I(h) and shifted the half activation voltage (V(half)) to more positive values (-104 +/- 3 to -91 +/- 2 mV). Blocking I(h) with 50 microM ZD7288 resulted in hyperpolarization of the resting membrane potential (approximately 4 mV) and slowing the decay of the EPSP by 47%, suggesting that I(h) is active at rest and shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first synapse in the auditory pathway.

  7. Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome

    NARCIS (Netherlands)

    Croxen, R; Newland, C; Beeson, D; Oosterhuis, H; Chauplannaz, G; Vincent, A; NewsomDavis, J

    1997-01-01

    Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine rece

  8. Effect of morphine on L-calcium channel of SK-N-SH human neuroblastoma cells%吗啡对人神经母细胞瘤SK-N-SH细胞L-Ca2+ 通道的作用

    Institute of Scientific and Technical Information of China (English)

    雷洪伊; 徐世元; 张庆国; 叶小平

    2008-01-01

    Objective To study the effect of morphine on L-calcium channels of SK-N-SH human neuroblastoma cells.Metllods SK-N-SH human neuroblastoma cells were cultured in vitro. Electrical current and conductance of calcium channels of SK-N-SH cells were measured using cell-attached patch clamp technique in the presence of morphine with different concentrations(0.10-8 mol/L,10-7 mol/L,10-6 mol/L and 10-5 mol/L)preteated with or without naloxone(10-5 mol/L).Results Electrical current of L-calcium channels of SK-N-SH human neuroblastoma cells was concentration-dependently depressed by morphine(P <0.01 at concentrations of 10-7,10-6 and 10-5 mol/L).Pretreatment with naloxone eliminated the morphine-induced depression on electrical current of calcium channels. Electric conductance of calcium channels did not change in the presence or absence of morohine. Conclusions Morphine inhibits electric current of L-type calcium channels of SK-N-SH human neuroblastoma cells in a manner of concentration dependence,probably via blockade of μ-opioid receptor;but does not influence electric conductence ot L-type calcium channels.%目的 观察吗啡对人神经母细胞瘤SK-N-SH细胞L-Ca2+通道的影响.方法 培养人神经母细胞瘤SK-N-SH细胞,应用细胞贴附式膜片钳技术记录在不同浓度吗啡(0、10-8 mol/L、10-7 mol/L、10-6 mol/L、10-5 mol/L)作用下SK-N-SH细胞L-Ca2+通道电流和电导变化.预先加入10-5 mol/L纳洛酮后,再依次加入上述不同浓度吗啡,记录此通道电流和电导.结果 10-7 mol/L、10-6 mol/L、10-5 mol/L吗啡可抑制SK-N-SH细胞L-型Ca2+通道电流,吗啡浓度为10-6mol/L、10-5 mol/L时抑制作用更明显(P<0.01),具有浓度依赖性;各浓度吗啡对L-Ca2+通道电导无影响;预先加入纳络酮,可阻断吗啡对该通道的抑制作用.结论 吗啡可抑制SK-N-SH细胞上L-Ca2+通道电流,对该通道电导无作用.纳络酮可翻转吗啡的作用,其抑制作用可能通过μ阿片受体产生.

  9. Funny current and cardiac rhythm: insights from HCN knockout mouse models

    Directory of Open Access Journals (Sweden)

    Mirko eBaruscotti

    2012-07-01

    Full Text Available In the adult animal the sinoatrial node (SAN rhythmically generates a depolarizing wave that propagates to the rest of the heart. However, the SAN is more than a simple clock; it is a clock that adjusts its pace according to the metabolic requirements of the organism. The Hyperpolarization-activated Cyclic Nucleotide-gated channels (HCN1-4 are the structural component of the funny (If channels; in the SAN the If current is the main driving electrical force of the diastolic depolarization and the HCN4 is the most abundant isoform. The generation of HCN KO mouse models has advanced the understanding of the role of these channels in cardiac excitability. The HCN4 KO models that were first developed allowed either global or cardiac-specific constitutive ablation of HCN4 channels, and resulted in embryonic lethality. A further progress was made with the development of three separate inducible HCN4 KO models; in one model KO was induced globally in the entire organism, in a second, ablation occurred only in HCN4-expressing cells, and finally in a third model KO was confined to cardiac cells. Unexpectedly, the three models yielded different results; similarities and differences among these models will be presented and discussed. The functional effects of HCN2 and HCN3 knockout models and transgenic HCN4 mouse models will also be discussed.In conclusion, HCN KO/transgenic models have allowed to evaluate the functional role of the If currents in intact animals as well as in single SAN cells isolated from the same animals. This opportunity is therefore unique since it allows to 1 verify the contribution of specific HCN isoforms to cardiac activity in intact animals, and 2 to compare these results to those obtained in single cell experiments. These combined studies were not possible prior to the development of KO models. Finally, these models represent critical tools to improve our understanding of the molecular basis of some inheritable arrhythmic human

  10. [Synopsis about the hypothesis of "information channel" of channel-collateral system].

    Science.gov (United States)

    Chang, Xi-Lang

    2008-10-01

    The author of the present paper founded a theorem about the "incompleteness of single channel structure" (nerve, blood vessel, lymphatic, interspace, aperture, etc.) through quantitative and qualitative analysis about the economic information channel in the human body, which eliminates the probability of single channel structure in the information channel of channel (meridian)-collateral system. After comprehensive analysis on the current researches, the author puts forward a neodoxy, i.e., the body "information channel" structure of the channel-collateral system, mainly follows the distribution regularity of systemic statistics, and is not a single specific entity; various layers of the information channel in the main stems of the channel-collaterals are composed of optimized structure tissues. Hence, the structure of this information channel of channel-collateral system is an overall-optimized, sequential and compatible systemic structure. From this neodoxy, the author brings forward a working principle of channel-collaterals, which is supported theoretically by bio-auxology. The longitudinal distribution of the main stems of meridian-collaterals is considered to result from that in the process of the animal evolution, in the animals moving forward, the microscopic complicated movement of intracorporeal information and energy molecules is related to the forward macroscopic and non-uniform movement of organism in trans-measure. Its impulse and kinetic momentum forms a main vector in the longitudinal direction of the body (the direction of the main stem of channel-collaterals). In order to adapt to and utilize natural regularities, the main stems of the channel-collaterals gradually differentiate and evolve in the living organism, forming a whole system. The "hypothesis of biological origin of channel-collateral system" and "that of information channel of the channel-collaterals in the body" constitute a relatively complete theoretical system framework.

  11. Solution structures of the cytoplasmic linkers between segments S4 and S5 (S4-S5) in domains III and IV of human brain sodium channels in SDS micelles.

    Science.gov (United States)

    Miyamoto, K; Nakagawa, T; Kuroda, Y

    2001-09-01

    The two cytoplasmic linkers connecting segment S4 and segment S5 (S4-S5 linker) of both domain III (III/S4-S5) and IV (IV/S4-S5) of the sodium channel alpha-subunit are considered to work as a hydrophobic receptor for the inactivation particle because of the three hydrophobic amino acids of Ile-Phe-Met (IFM motif) in the III-IV linker of the sodium channel alpha-subunit. To date, the solution structures of the peptides related to III/S4-S5 (MP-D3: A1325-M1338) and IV/S4-S5 (MP-D4: T1648-L1666) of human brain sodium channels have been investigated using CD and (1)H NMR spectroscopies. SDS micelles were employed as a solvent. The micelles mimic either biological membranes or the interior of a protein and can be a relevant environment at the inactivated state of the channels. It was found that the secondary structures of both MP-D3 and MP-D4 assume alpha-helical conformations around the N-terminal half-side of the sequences, i.e. the residues between V1326 and L1331 in MP-D3 and between L1650 and S1656 in MP-D4. Residue A1329 in MP-D3, which is considered to interact with F1489 of the IFM motif, was found to be located within the alpha-helix. Residues F1651, M1654, M1655, L1657 and A1669 in MP-D4, which also play an important role in inactivation, formed a hydrophobic cluster on one side of the helix. This cluster was concluded to interact with the hydrophobic cluster due to the III-IV linker before the inactivation gate closes.

  12. Mobile radio channels

    CERN Document Server

    Pätzold, Matthias

    2011-01-01

    Providing a comprehensive overview of the modelling, analysis and simulation of mobile radio channels, this book gives a detailed understanding of fundamental issues and examines state-of-the-art techniques in mobile radio channel modelling. It analyses several mobile fading channels, including terrestrial and satellite flat-fading channels, various types of wideband channels and advanced MIMO channels, providing a fundamental understanding of the issues currently being investigated in the field. Important classes of narrowband, wideband, and space-time wireless channels are explored in deta

  13. Channel nut tool

    Energy Technology Data Exchange (ETDEWEB)

    Olson, Marvin

    2016-01-12

    A method, system, and apparatus for installing channel nuts includes a shank, a handle formed on a first end of a shank, and an end piece with a threaded shaft configured to receive a channel nut formed on the second end of the shaft. The tool can be used to insert or remove a channel nut in a channel framing system and then removed from the channel nut.

  14. Plectasin, First Animal Toxin-Like Fungal Defensin Blocking Potassium Channels through Recognizing Channel Pore Region

    Directory of Open Access Journals (Sweden)

    Fang Xiang

    2015-01-01

    Full Text Available The potassium channels were recently found to be inhibited by animal toxin-like human β-defensin 2 (hBD2, the first defensin blocker of potassium channels. Whether there are other defensin blockers from different organisms remains an open question. Here, we reported the potassium channel-blocking plectasin, the first defensin blocker from a fungus. Based on the similar cysteine-stabilized alpha-beta (CSαβ structure between plectasin and scorpion toxins acting on potassium channels, we found that plectasin could dose-dependently block Kv1.3 channel currents through electrophysiological experiments. Besides Kv1.3 channel, plectasin could less inhibit Kv1.1, Kv1.2, IKCa, SKCa3, hERG and KCNQ channels at the concentration of 1 μΜ. Using mutagenesis and channel activation experiments, we found that outer pore region of Kv1.3 channel was the binding site of plectasin, which is similar to the interacting site of Kv1.3 channel recognized by animal toxin blockers. Together, these findings not only highlight the novel function of plectasin as a potassium channel inhibitor, but also imply that defensins from different organisms functionally evolve to be a novel kind of potassium channel inhibitors.

  15. NALCN ion channels have alternative selectivity filters resembling calcium channels or sodium channels.

    Directory of Open Access Journals (Sweden)

    Adriano Senatore

    Full Text Available NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE or sodium channels (EKEE or EEKE. NALCN channels with alternative calcium, (EEEE and sodium, (EKEE or EEKE -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG(+ impermeant and blockable with 10 µM Gd(3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2:371-83.

  16. Structure and functional dynamics characterization of the ion channel of the human respiratory syncytial virus (hRSV) small hydrophobic protein (SH) transmembrane domain by combining molecular dynamics with excited normal modes.

    Science.gov (United States)

    Araujo, Gabriela C; Silva, Ricardo H T; Scott, Luis P B; Araujo, Alexandre S; Souza, Fatima P; de Oliveira, Ronaldo Junio

    2016-12-01

    The human respiratory syncytial virus (hRSV) is the major cause of lower respiratory tract infection in children and elderly people worldwide. Its genome encodes 11 proteins including SH protein, whose functions are not well known. Studies show that SH protein increases RSV virulence degree and permeability to small compounds, suggesting it is involved in the formation of ion channels. The knowledge of SH structure and function is fundamental for a better understanding of its infection mechanism. The aim of this study was to model, characterize, and analyze the structural behavior of SH protein in the phospholipids bilayer environment. Molecular modeling of SH pentameric structure was performed, followed by traditional molecular dynamics (MD) simulations of the protein immersed in the lipid bilayer. Molecular dynamics with excited normal modes (MDeNM) was applied in the resulting system in order to investigate long time scale pore dynamics. MD simulations support that SH protein is stable in its pentameric form. Simulations also showed the presence of water molecules within the bilayer by density distribution, thus confirming that SH protein is a viroporin. This water transport was also observed in MDeNM studies with histidine residues of five chains (His22 and His51), playing a key role in pore permeability. The combination of traditional MD and MDeNM was a very efficient protocol to investigate functional conformational changes of transmembrane proteins that act as molecular channels. This protocol can support future investigations of drug candidates by acting on SH protein to inhibit viral infection. Graphical Abstract The ion channel of the human respiratory syncytial virus (hRSV) small hydrophobic protein (SH) transmembrane domainᅟ.

  17. Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

    Science.gov (United States)

    La Fuente, José M; Fernández, Argentina; Cuevas, Pedro; González-Corrochano, Rocío; Chen, Mao Xiang; Angulo, Javier

    2014-07-15

    We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.

  18. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    Science.gov (United States)

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

  19. Multi-Channel Retailing

    Directory of Open Access Journals (Sweden)

    Dirk Morschett, Dr.,

    2005-01-01

    Full Text Available Multi-channel retailing entails the parallel use by retailing enterprises of several sales channels. The results of an online buyer survey which has been conducted to investigate the impact of multi-channel retailing (i.e. the use of several retail channels by one retail company on consumer behaviour show that the frequently expressed concern that the application of multi-channel systems in retailing would be associated with cannibalization effects, has proven unfounded. Indeed, the appropriate degree of similarity, consistency, integration and agreement achieves the exact opposite. Different channels create different advantages for consumers. Therefore the total benefit an enterprise which has a multi-channel system can offer to its consumers is larger, the greater the number of available channels. The use of multi-channel systems is associated with additional purchases in the different channels. Such systems are thus superior to those offering only one sales channel to their customers. Furthermore, multi-channel systems with integrated channels are superior to those in which the channels are essentially autonomous and independent of one another. In integrated systems, consumers can achieve synergy effects in the use of sales-channel systems. Accordingly, when appropriately formulated, multi-channel systems in retailing impact positively on consumers. They use the channels more frequently, buy more from them and there is a positive customer-loyalty impact. Multi-channel systems are strategic options for achieving customer loyalty, exploiting customer potential and for winning new customers. They are thus well suited for approaching differing and varied target groups.

  20. An Upregulation in the Expression of Vanilloid Transient Potential Channels 2 Enhances Hypotonicity-Induced Cytosolic Ca2+ Rise in Human Induced Pluripotent Stem Cell Model of Hutchinson Gillford Progeria

    Science.gov (United States)

    Ho, Jenny Chung-Yee; Siu, Chung-Wah; Cheung, Sin-Ying; Tang, Nelson L.; Yu, Shan; Tse, Hung-Fat; Yao, Xiaoqiang

    2014-01-01

    Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca2+ ([Ca2+]i) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca2+]i rise in iPSC-ECs from normal individuals but a sustained [Ca2+]i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca2+]i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca2+]i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca2+]i elevation in HGPS-iPSC-ECs under hypotonicity, consequently

  1. An upregulation in the expression of vanilloid transient potential channels 2 enhances hypotonicity-induced cytosolic Ca²⁺ rise in human induced pluripotent stem cell model of Hutchinson-Gillford Progeria.

    Directory of Open Access Journals (Sweden)

    Chun-Yin Lo

    Full Text Available Hutchinson-Gillford Progeria Syndrome (HGPS is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90 iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²⁺ ([Ca²⁺](i rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²⁺](i rise in iPSC-ECs from normal individuals but a sustained [Ca²⁺](i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM, and a specific TRPV2 channel inhibitor, tranilast (100 µM, abolished the sustained phase of hypotonicity-induced [Ca²⁺](i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²⁺](i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²⁺](i elevation in HGPS

  2. An upregulation in the expression of vanilloid transient potential channels 2 enhances hypotonicity-induced cytosolic Ca²⁺ rise in human induced pluripotent stem cell model of Hutchinson-Gillford Progeria.

    Science.gov (United States)

    Lo, Chun-Yin; Tjong, Yung-Wui; Ho, Jenny Chung-Yee; Siu, Chung-Wah; Cheung, Sin-Ying; Tang, Nelson L; Yu, Shan; Tse, Hung-Fat; Yao, Xiaoqiang

    2014-01-01

    Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²⁺ ([Ca²⁺](i)) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²⁺](i) rise in iPSC-ECs from normal individuals but a sustained [Ca²⁺](i) elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca²⁺](i) rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²⁺](i) rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²⁺](i) elevation in HGPS

  3. Glutamine substitution at alanine1649 in the S4-S5 cytoplasmic loop of domain 4 removes the voltage sensitivity of fast inactivation in the human heart sodium channel.

    Science.gov (United States)

    Tang, L; Chehab, N; Wieland, S J; Kallen, R G

    1998-05-01

    Normal activation-inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4-S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89-104.). We now show that glutamine substitutions NH2-terminal to the methionines (L1646, L1647, F1648, A1649, L1650) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4-S5/D4 segment (L1654, P1655, A1656) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641-658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A1649 or Y1494Y1495 may disrupt a linkage between S4-S5/D4 and the interdomain 3-4 loop interfering with normal activation-inactivation coupling.

  4. River channel adjustments in Southern Italy over the past 150 years and implications for channel recovery

    Science.gov (United States)

    Scorpio, Vittoria; Aucelli, Pietro P. C.; Giano, Salvatore I.; Pisano, Luca; Robustelli, Gaetano; Rosskopf, Carmen M.; Schiattarella, Marcello

    2015-12-01

    Multi-temporal GIS analysis of topographic maps and aerial photographs along with topographic and geomorphological surveys are used to assess evolutionary trends and key control factors of channel adjustments for five major rivers in southern Italy (the Trigno, Biferno, Volturno, Sinni and Crati rivers) to support assessment of channel recovery and river restoration. Three distinct phases of channel adjustment are identified over the past 150 years primarily driven by human disturbances. Firstly, slight channel widening dominated from the last decades of the nineteenth century to the 1950s. Secondly, from the 1950s to the end of the 1990s, altered sediment fluxes induced by in-channel mining and channel works brought about moderate to very intense incision (up to 6-7 m) accompanied by strong channel narrowing (up to 96%) and changes in channel configuration from multi-threaded to single-threaded patterns. Thirdly, the period from around 2000 to 2015 has been characterized by channel stabilization and local widening. Evolutionary trajectories of the rivers studied are quite similar to those reconstructed for other Italian rivers, particularly regarding the second phase of channel adjustments and ongoing transitions towards channel recovery in some reaches. Analyses of river dynamics, recovery potential and connectivity with sediment sources of the study reaches, framed in their catchment context, can be used as part of a wider interdisciplinary approach that views effective river restoration alongside sustainable and risk-reduced river management.

  5. Hadamard quantum broadcast channels

    Science.gov (United States)

    Wang, Qingle; Das, Siddhartha; Wilde, Mark M.

    2017-10-01

    We consider three different communication tasks for quantum broadcast channels, and we determine the capacity region of a Hadamard broadcast channel for these various tasks. We define a Hadamard broadcast channel to be such that the channel from the sender to one of the receivers is entanglement-breaking and the channel from the sender to the other receiver is complementary to this one. As such, this channel is a quantum generalization of a degraded broadcast channel, which is well known in classical information theory. The first communication task we consider is classical communication to both receivers, the second is quantum communication to the stronger receiver and classical communication to other, and the third is entanglement-assisted classical communication to the stronger receiver and unassisted classical communication to the other. The structure of a Hadamard broadcast channel plays a critical role in our analysis: The channel to the weaker receiver can be simulated by performing a measurement channel on the stronger receiver's system, followed by a preparation channel. As such, we can incorporate the classical output of the measurement channel as an auxiliary variable and solve all three of the above capacities for Hadamard broadcast channels, in this way avoiding known difficulties associated with quantum auxiliary variables.

  6. USACE Navigation Channels 2012

    Data.gov (United States)

    California Department of Resources — This dataset represents both San Francisco and Los Angeles District navigation channel lines. All San Francisco District channel lines were digitized from CAD files...

  7. Channelling versus inversion

    DEFF Research Database (Denmark)

    Gale, A.S.; Surlyk, Finn; Anderskouv, Kresten

    2013-01-01

    . Within this channel were smaller erosional structures (hardgrounds, and locally have a basal fill of granular phosphorite. The entire channel system was progressively infilled by chalk, as demonstrated by the expanded succession...

  8. Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).

    Science.gov (United States)

    Samways, Damien S K; Khakh, Baljit S; Dutertre, Sébastien; Egan, Terrance M

    2011-08-16

    P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.

  9. SwissProt search result: AK099993 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099993 J013132D09 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 5e-17 ...

  10. SwissProt search result: AK242807 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242807 J090060H17 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 3e-28 ...

  11. SwissProt search result: AK067626 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067626 J013112I06 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 4e-11 ...

  12. SwissProt search result: AK069229 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069229 J023012H21 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 9e-16 ...

  13. SwissProt search result: AK120308 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120308 J013054N15 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 2e-20 ...

  14. SwissProt search result: AK243602 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243602 J100084P18 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 3e-20 ...

  15. SwissProt search result: AK100739 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100739 J023118D04 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 7e-29 ...

  16. SwissProt search result: AK100808 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100808 J023121M17 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 4e-36 ...

  17. SwissProt search result: AK100312 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100312 J023078M02 (Q9TV66) Potassium/sodium hyperpolarization-activated cyclic nu...cleotide-gated channel 4 (Hyperpolarization-activated cation channel 4) (HAC-4) HCN4_RABIT 6e-14 ...

  18. Quantum Multiple Access Channel

    Institute of Scientific and Technical Information of China (English)

    侯广; 黄民信; 张永德

    2002-01-01

    We consider the transmission of classical information over a quantum channel by many senders, which is a generalization of the two-sender case. The channel capacity region is shown to be a convex hull bound by the yon Neumann entropy and the conditional yon Neumann entropies. The result allows a reasonable distribution of channel capacity over the senders.

  19. Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

    Science.gov (United States)

    Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

    2017-02-10

    Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Ion channels in plants

    Science.gov (United States)

    Baluška, František; Mancuso, Stefano

    2013-01-01

    In his recent opus magnum review paper published in the October issue of Physiology Reviews, Rainer Hedrich summarized the field of plant ion channels.1 He started from the earliest electric recordings initiated by Charles Darwin of carnivorous Dionaea muscipula,1,2 known as Venus flytrap, and covered the topic extensively up to the most recent discoveries on Shaker-type potassium channels, anion channels of SLAC/SLAH families, and ligand-activated channels of glutamate receptor-like type (GLR) and cyclic nucleotide-gated channels (CNGC).1 PMID:23221742

  1. Establishment of human sperm-specific voltage-dependent anion channel 3 recombinant vector for the production of a male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Asmarinah Asmarinah

    2012-05-01

    Full Text Available Background: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine.Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp. E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene.Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene.Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine. (Med J Indones. 2012;21:61-5Keywords: Contraception, recombinant vector, sperm, VDAC3

  2. Improved functional expression of recombinant human ether-a-go-go (hERG K+ channels by cultivation at reduced temperature

    Directory of Open Access Journals (Sweden)

    Hamilton Bruce

    2007-12-01

    Full Text Available Abstract Background HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance. Results Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C. Conclusion Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.

  3. Protocol channels as a new design alternative of covert channels

    CERN Document Server

    Wendzel, Steffen

    2008-01-01

    Covert channel techniques are used by attackers to transfer hidden data. There are two main categories of covert channels: timing channels and storage channels. This paper introduces a third category called protocol channels. A protocol channel switches one of at least two protocols to send a bit combination to a destination while sent packets include no hidden information themselves.

  4. Surface vacancy channels through ion channeling

    Energy Technology Data Exchange (ETDEWEB)

    Redinger, Alex; Standop, Sebastian; Michely, Thomas [II. Physikalisches Institut, Universitaet Koeln, Zuelpicher Strasse 77, 50937 Koeln (Germany); Rosandi, Yudi; Urbassek, Herbert M. [Fachbereich Physik, Technische Universitaet Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern (Germany)

    2009-07-01

    Damage patterns of single ion impacts on Pt(111) have been studied by scanning tunneling microscopy (STM) and molecular dynamics simulations (MD). Low temperature experiments, where surface diffusion is absent, have been performed for argon and xenon ions with energies between 1 keV and 15 keV at an angle of incidence of 86 {sup circle} measured with respect to the surface normal. Ions hitting preexisting illuminated step edges penetrate into the crystal and are guided in open crystallographic directions, one or more layers underneath the surface (subsurface channeling). In the case of argon channeling the resulting surface damage consists of adatom and vacancy pairs aligned in ion beam direction. After xenon channeling thin surface vacancy trenches along the ion trajectories - surface vacancy channels - are observed. They result from very efficient sputtering and adatom production along the ion trajectory. This phenomena is well reproduced in molecular dynamics simulations of single ion impacts at 0 K. The damage patterns of Argon and Xenon impacts can be traced back to the different energy losses of the particles in the channel. Channeling distances exceeding 1000 A for 15 keV xenon impacts are observed.

  5. Calcium channel blockers and Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Yi Tan; Yulin Deng; Hong Qing

    2012-01-01

    Alzheimer's disease is characterized by two pathological hallmarks: amyloid plaques and neurofi-brillary tangles. In addition, calcium homeostasis is disrupted in the course of human aging. Recent research shows that dense plaques can cause functional alteration of calcium signals in mice with Alzheimer's disease. Calcium channel blockers are effective therapeutics for treating Alzheimer's disease. This review provides an overview of the current research of calcium channel blockers in-volved in Alzheimer's disease therapy.

  6. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    Science.gov (United States)

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

  7. Superior spatial resolution in confocal X-ray techniques using collimating channel array optics: elemental mapping and speciation in archaeological human bone

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, S.; Agyeman-Budu, D. N.; Woll, A. R.; Swanston, T.; Varney, T. L.; Cooper, D. M. L.; Hallin, E.; George, G. N.; Pickering, I. J.; Coulthard, I.

    2017-01-01

    Confocal X-ray fluorescence imaging (CXFI) and confocal X-ray absorption spectroscopy (CXAS) respectively enable the study of three dimensionally resolved localization and speciation of elements. Applied to a thick sample, essentially any volume element of interest within the X-ray fluorescence escape depth can be examined without the need for physical thin sectioning. To date, X-ray confocal detection generally has employed a polycapillary optic in front of the detector to collect fluorescence from the probe volume formed at the intersection of its focus with the incident microfocus beam. This work demonstrates the capability of a novel Collimating Channel Array (CCA) optic in providing an improved and essentially energy independent depth resolution approaching 2 μm. By presenting a comparison of elemental maps of archaeological bone collected without confocal detection, and with polycapillary- and CCA-based confocal detection, this study highlights the strengths and limitations of each mode. Unlike the polycapillary, the CCA shows similar spatial resolution in maps for both low (Ca) and high (Pb and Sr) energy X-ray fluorescence, thus illustrating the energy independent nature of the CCA optic resolution. While superior spatial resolution is demonstrated for all of these elements, the most significant improvement is observed for Ca, demonstrating the advantage of employing the CCA optic in examining light elements. In addition to CXFI, this configuration also enables the collection of Pb L3 CXAS data from micro-volumes with dimensions comparable to bone microstructures of interest. Our CXAS result, which represents the first CCA-based biological CXAS, demonstrates the ability of CCA optics to collect site specific spectroscopic information. The demonstrated combination of site-specific elemental localization and speciation data will be useful in diverse fields.

  8. Quantum broadcast channels

    CERN Document Server

    Yard, J; Devetak, I; Yard, Jon; Hayden, Patrick; Devetak, Igor

    2006-01-01

    We analyze quantum broadcast channels, which are quantum channels with a single sender and many receivers. Focusing on channels with two receivers for simplicity, we generalize a number of results from the network Shannon theory literature which give the rates at which two senders can receive a common message, while a personalized one is sent to one of them. Our first collection of results applies to channels with a classical input and quantum outputs. The second class of theorems we prove concern sending a common classical message over a quantum broadcast channel, while sending quantum information to one of the receivers. The third group of results we obtain concern communication over an isometry, giving the rates at quantum information can be sent to one receiver, while common quantum information is sent to both, in the sense that tripartite GHZ entanglement is established. For each scenario, we provide an additivity proof for an appropriate class of channels, yielding single-letter characterizations of the...

  9. Volume Regulated Channels

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjær

    of volume perturbations evolution have developed system of channels and transporters to tightly control volume homeostasis. In the past decades evidence has been mounting, that the importance of these volume regulated channels and transporters are not restricted to the defense of cellular volume...... but are also essential for a number of physiological processes such as proliferation, controlled cell death, migration and endocrinology. The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and the transient receptor potential Vanilloid (TRPV4) channel. I: Cl......- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cell volume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a major potential of regulating cellular function. These functions include control of the cell cycle...

  10. Increased store-operated and 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx in monocytes is mediated by transient receptor potential canonical channels in human essential hypertension

    DEFF Research Database (Denmark)

    Liu, Dao Yan; Thilo, Florian; Scholze, Alexandra;

    2007-01-01

    Activation of nonselective cation channels of the transient receptor potential canonical (TRPC) family has been associated with hypertension. Whether store-operated channels, which are activated after depletion of intracellular stores, or second-messenger-operated channels, which are activated by 1......-oleoyl-2-acetyl-sn-glycerol, are affected in essential hypertension is presently unknown....

  11. 基于多通道信息融合的双人交互动作识别算法%Two-human Interaction Recognition Algorithm Based on Multi-channels Information Fusion

    Institute of Scientific and Technical Information of China (English)

    黄菲菲; 曹江涛; 姬晓飞

    2016-01-01

    基于视频的双人交互行为识别是计算机视觉领域中一个富有挑战性的研究课题。针对基于整体的双人交互动作识别方法的特征表示复杂度高及匹配方法难以确定的问题,文中提出了一种基于多通道信息融合的双人交互动作识别算法。该方法首先采用更符合人类视觉系统的HSI颜色空间模型,分别通过H、S、I三个通道来提取HOG特征并进行直方图统计表示,使用最近邻分类器分别获得三通道下的识别结果,然后对识别结果进行等比例融合得到待测视频的最终识别结果。该方法在UT-interaction上进行了测试,得到了81.7%的识别率,证明了该方法的有效性及可行性。将其与相同数据库下的其他方法进行比较,结果表明该方法特征易于提取,计算效率高,避免了复杂的运算,具有一定的应用价值。%Two-human interaction recognition based on video is a challenging research topic in computer vision. Aiming at the problem of high complexity for feature representation and matching method hard to determine for the two-human interaction recognition method,a two-human interaction recognition algorithm based on multi-channels information fusion is proposed in this paper. Firstly,HSI color space model which is more fit for the human visual system is used. Respectively by H,S,I three channels to extract the HOG feature for histogram statistics representation,the nearest neighbor classifier is used to require the identification results respectively under the three-channel,then the results is integrated with equal ratio to obtain the overall recognition rate. The proposed method is tested on UT-interac-tion which has achieved recognition ratio of 81. 7%,proving the validity and feasibility of this method. Compared with other methods,the proposed method has higher calculation efficiency and recognition accuracy with increasing number of potential applications.

  12. Ion channels in asthma.

    Science.gov (United States)

    Valverde, Miguel A; Cantero-Recasens, Gerard; Garcia-Elias, Anna; Jung, Carole; Carreras-Sureda, Amado; Vicente, Rubén

    2011-09-23

    Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.

  13. Quantum feedback channels

    CERN Document Server

    Bowen, G

    2002-01-01

    In classical information theory the capacity of a noisy communication channel cannot be increased by the use of feedback. In quantum information theory the no-cloning theorem means that noiseless copying and feedback of quantum information cannot be achieved. In this paper, quantum feedback is defined as the unlimited use of a noiseless quantum channel from receiver to sender. Given such quantum feedback, it is shown to provide no increase in the entanglement-assisted capacities of a noisy quantum channel, in direct analogy to the classical case. It is also shown that in various cases of non-assisted capacities, feedback can increase the capacity of many quantum channels.

  14. TRP channels in schistosomes

    Directory of Open Access Journals (Sweden)

    Swarna Bais

    2016-12-01

    Full Text Available Praziquantel (PZQ is effectively the only drug currently available for treatment and control of schistosomiasis, a disease affecting hundreds of millions of people worldwide. Many anthelmintics, likely including PZQ, target ion channels, membrane protein complexes essential for normal functioning of the neuromusculature and other tissues. Despite this fact, only a few classes of parasitic helminth ion channels have been assessed for their pharmacological properties or for their roles in parasite physiology. One such overlooked group of ion channels is the transient receptor potential (TRP channel superfamily. TRP channels share a common core structure, but are widely diverse in their activation mechanisms and ion selectivity. They are critical to transducing sensory signals, responding to a wide range of external stimuli. They are also involved in other functions, such as regulating intracellular calcium and organellar ion homeostasis and trafficking. Here, we review current literature on parasitic helminth TRP channels, focusing on those in schistosomes. We discuss the likely roles of these channels in sensory and locomotor activity, including the possible significance of a class of TRP channels (TRPV that is absent in schistosomes. We also focus on evidence indicating that at least one schistosome TRP channel (SmTRPA has atypical, TRPV1-like pharmacological sensitivities that could potentially be exploited for future therapeutic targeting.

  15. KV7 potassium channels

    DEFF Research Database (Denmark)

    Stott, Jennifer B; Jepps, Thomas Andrew; Greenwood, Iain A

    2014-01-01

    identified as being crucial mediators of this process in a variety of smooth muscle. Recently, KV7 channels have been shown to be involved in the pathogenesis of hypertension, as well as being implicated in other smooth muscle disorders, providing a new and inviting target for smooth muscle disorders.......Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been...

  16. Dysfunctional HCN ion channels in neurological diseases.

    Science.gov (United States)

    DiFrancesco, Jacopo C; DiFrancesco, Dario

    2015-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed as four different isoforms (HCN1-4) in the heart and in the central and peripheral nervous systems. HCN channels are activated by membrane hyperpolarization at voltages close to resting membrane potentials and carry the hyperpolarization-activated current, dubbed If (funny current) in heart and Ih in neurons. HCN channels contribute in several ways to neuronal activity and are responsible for many important cellular functions, including cellular excitability, generation, and modulation of rhythmic activity, dendritic integration, transmission of synaptic potentials, and plasticity phenomena. Because of their role, defective HCN channels are natural candidates in the search for potential causes of neurological disorders in humans. Several data, including growing evidence that some forms of epilepsy are associated with HCN mutations, support the notion of an involvement of dysfunctional HCN channels in different experimental models of the disease. Additionally, some anti-epileptic drugs are known to modify the activity of the Ih current. HCN channels are widely expressed in the peripheral nervous system and recent evidence has highlighted the importance of the HCN2 isoform in the transmission of pain. HCN channels are also present in the midbrain system, where they finely regulate the activity of dopaminergic neurons, and a potential role of these channels in the pathogenesis of Parkinson's disease has recently emerged. The function of HCN channels is regulated by specific accessory proteins, which control the correct expression and modulation of the neuronal Ih current. Alteration of these proteins can severely interfere with the physiological channel function, potentially predisposing to pathological conditions. In this review we address the present knowledge of the association between HCN dysfunctions and neurological diseases, including clinical, genetic, and physiopathological

  17. Dysfunctional HCN ion channels in neurological diseases

    Directory of Open Access Journals (Sweden)

    Jacopo C. DiFrancesco

    2015-03-01

    Full Text Available Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are expressed as four different isoforms (HCN1-4 in the heart and in the central and peripheral nervous systems. HCN channels are activated by membrane hyperpolarization at voltages close to resting membrane potentials and carry the hyperpolarization-activated current, dubbed If (funny current in heart and Ih in neurons. HCN channels contribute in several ways to neuronal activity and are responsible for many important cellular functions, including cellular excitability, generation and modulation of rhythmic activity, dendritic integration, transmission of synaptic potentials and plasticity phenomena. Because of their role, defective HCN channels are natural candidates in the search for potential causes of neurological disorders in humans. Several data, including growing evidence that some forms of epilepsy are associated with HCN mutations, support the notion of an involvement of dysfunctional HCN channels in different experimental models of the disease. Additionally, some anti-epileptic drugs are known to modify the activity of the Ih current. HCN channels are widely expressed in the peripheral nervous system and recent evidence has highlighted the importance of the HCN2 isoform in the transmission of pain. HCN channels are also present in the midbrain system, where they finely regulate the activity of dopaminergic neurons, and a potential role of these channels in the pathogenesis of Parkinson’s disease has recently emerged. The function of HCN channels is regulated by specific accessory proteins, which control the correct expression and modulation of the neuronal Ih current. Alteration of these proteins can severely interfere with the physiological channel function, potentially predisposing to pathological conditions. In this review we address the present knowledge of the association between HCN dysfunctions and neurological diseases, including clinical, genetic and

  18. Examination of metabolic pathways and identification of human liver cytochrome P450 isozymes responsible for the metabolism of barnidipine, a calcium channel blocker.

    Science.gov (United States)

    Teramura, T; Fukunaga, Y; Van Hoogdalem, E J; Watanabe, T; Higuchi, S

    1997-09-01

    1. In a human liver microsomal system, barnidipine was converted into three primary metabolites, an N-debenzylated product (M-1), a hydrolyzed product of the benzyl-pyrrolidine ester (M-3) and an oxidized product of the dihydropyridine ring (M-8). 2. Involvement of CYP3A in the three primary metabolic pathways was revealed by the following studies: (a) inhibition of CYP3A, (b) a correlation study using 10 individual human liver microsomes and (c) cDNA-expression studies. The secondary metabolites, M-2 and M-4 (pyridine forms of M-1 and M-3), were most likely generated from M-8 but were unlikely from M-1 or M-3. Involvement of CYP3A in the secondary pathways of metabolism is also suggested. 3. The possibility of interactions between barnidipine and coadministered drugs was examined in vitro. The formation rate of the primary metabolites was little affected by warfarin, theophylline, phenytoin, diclofenac and amitriptyline at concentrations of 200 microM, but was inhibited by glibenclamide, simvastatin and cyclosporin A. IC50 for the latter drugs was estimated to be > 200, 200 and 20 microM respectively, which was roughly > 200, 6000 and 50 times higher than their respective therapeutic plasma levels, suggesting that interactions with cyclosporin A, a CYP3A inhibitor, are of possible clinical relevance.

  19. Co-localisation of K(ir)4.1 and AQP4 in rat and human cochleae reveals a gap in water channel expression at the transduction sites of endocochlear K(+) recycling routes.

    Science.gov (United States)

    Eckhard, Andreas; Gleiser, Corinna; Rask-Andersen, Helge; Arnold, Heinz; Liu, Wei; Mack, Andreas; Müller, Marcus; Löwenheim, Hubert; Hirt, Bernhard

    2012-10-01

    Sensory transduction in the cochlea depends on perilymphatic-endolymphatic potassium (K(+)) recycling. It has been suggested that the epithelial supporting cells (SCs) of the cochlear duct may form the intracellular K(+) recycling pathway. Thus, they must be endowed with molecular mechanisms that facilitate K(+) uptake and release, along with concomitant osmotically driven water movements. As yet, no molecules have been described that would allow for volume-equilibrated transepithelial K(+) fluxes across the SCs. This study describes the subcellular co-localisation of the K(ir)4.1 K(+) channel (K(ir)4.1) and the aquaporin-4 water channel (AQP4) in SCs, on the basis of immunohistochemical double-labelling experiments in rat and human cochleae. The results of this study reveal the expression of K(ir)4.1 in the basal or basolateral membranes of the SCs in the sensory domain of the organ of Corti that are adjacent to hair cells and in the non-sensory domains of the inner and outer sulci that abut large extracellular fluid spaces. The SCs of the inner sulcus (interdental cells, inner sulcus cells) and the outer sulcus (Hensen's cells, outer sulcus cells) display the co-localisation of K(ir)4.1 and AQP4 expression. However, the SCs in the sensory domain of the organ of Corti reveal a gap in the expression of AQP4. The outer pillar cell is devoid of both K(ir)4.1 and AQP4. The subcellular co-localisation of K(ir)4.1 and AQP4 in the SCs of the cochlea described in this study resembles that of the astroglia of the central nervous system and the glial Mueller cells in the retina.

  20. Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid

    Directory of Open Access Journals (Sweden)

    Kisaalita WS

    2013-01-01

    Full Text Available Ze-Zhi Wu,1 Zheng-Wei Wang,1 Li-Guang Zhang,1 Zhi-Xing An,1 Dong-Huo Z