New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

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Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G L

2013-01-01

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage...... a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X...... precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated...

Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.

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Gonzalo E Yévenes

Full Text Available Glycine receptors (GlyRs are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA are positive modulators of α(1, α(2 and α(3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly potentiate α(1 GlyRs but inhibit α(2 and α(3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM region 2 and intracellular lysine 385 determine the positive modulation of α(1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α(2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α(1 GlyRs, without affecting inhibition of α(2 and α(3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

4-Chloropropofol enhances chloride currents in human hyperekplexic and artificial mutated glycine receptors

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de la Roche Jeanne

2012-09-01

Full Text Available Abstract Background The mammalian neurological disorder hereditary hyperekplexia can be attributed to various mutations of strychnine sensitive glycine receptors. The clinical symptoms of “startle disease” predominantly occur in the newborn leading to convulsive hypertonia and an exaggerated startle response to unexpected mild stimuli. Amongst others, point mutations R271Q and R271L in the α1-subunit of strychnine sensitive glycine receptors show reduced glycine sensitivity and cause the clinical symptoms of hyperekplexia. Halogenation has been shown to be a crucial structural determinant for the potency of a phenolic compound to positively modulate glycine receptor function. The aim of this in vitro study was to characterize the effects of 4-chloropropofol (4-chloro-2,6-dimethylphenol at four glycine receptor mutations. Methods Glycine receptor subunits were expressed in HEK 293 cells and experiments were performed using the whole-cell patch-clamp technique. Results 4-chloropropofol exerted a positive allosteric modulatory effect in a low sub-nanomolar concentration range at the wild type receptor (EC50 value of 0.08 ± 0.02 nM and in a micromolar concentration range at the mutations (1.3 ± 0.6 μM, 0.1 ± 0.2 μM, 6.0 ± 2.3 μM and 55 ± 28 μM for R271Q, L, K and S267I, respectively. Conclusions 4-chloropropofol might be an effective compound for the activation of mutated glycine receptors in experimental models of startle disease.

[Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

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Boronovskiĭ, S E; Nartsissov, Ia R

2009-01-01

Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

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Florian Wegner

Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

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Xuanmao Chen

Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

Potentiation of glycine-gated NR1/NR3A NMDA receptors relieves Ca2+-dependent outward rectification

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Christian Madry

2010-03-01

Full Text Available Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named excitatory glycine receptors. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I-V dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I-V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I-V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors.

In Vivo Protection against Strychnine Toxicity in Mice by the Glycine Receptor Agonist Ivermectin

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Ahmed Maher

2014-01-01

Full Text Available The inhibitory glycine receptor, a ligand-gated ion channel that mediates fast synaptic inhibition in mammalian spinal cord and brainstem, is potently and selectively inhibited by the alkaloid strychnine. The anthelminthic and anticonvulsant ivermectin is a strychnine-independent agonist of spinal glycine receptors. Here we show that ivermectin is an effective antidote of strychnine toxicity in vivo and determine time course and extent of ivermectin protection. Mice received doses of 1 mg/kg and 5 mg/kg ivermectin orally or intraperitoneally, followed by an intraperitoneal strychnine challenge (2 mg/kg. Ivermectin, through both routes of application, protected mice against strychnine toxicity. Maximum protection was observed 14 hours after ivermectin administration. Combining intraperitoneal and oral dosage of ivermectin further improved protection, resulting in survival rates of up to 80% of animals and a significant delay of strychnine effects in up to 100% of tested animals. Strychnine action developed within minutes, much faster than ivermectin, which acted on a time scale of hours. The data agree with a two-compartment distribution of ivermectin, with fat deposits acting as storage compartment. The data demonstrate that toxic effects of strychnine in mice can be prevented if a basal level of glycinergic signalling is maintained through receptor activation by ivermectin.

[3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

International Nuclear Information System (INIS)

Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

1999-01-01

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Characterisation of the human NMDA receptor subunit NR3A glycine binding site

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Nilsson, A; Duan, J; Mo-Boquist, L-L

2007-01-01

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons

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Mengwen Qi

2018-02-01

Full Text Available Background/Aims: Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4 is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Methods: Whole-cell patch clamp recording was employed to record glycine-activated current (IGly and Western blot was conducted to assess GlyRs subunits protein expression. Results: Application of TRPV4 agonist (GSK1016790A or 5,6-EET increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047 and GlyR (strychnine, indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC (BIM II or D-sphingosine or calcium/calmodulin-dependent protein kinase II (CaMKII (KN-62 or KN-93 antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv. injected with GSK1016790A for 5 d. Conclusion: Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission.

[{sup 3}H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

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Benke, D.; Honer, M.; Mohler, H. [Institute of Pharmacology, ETH and University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland); Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C. [NS Research, Novartis Pharma AG, CH-4002 Basle (Switzerland)

1999-02-01

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [{sup 3}H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [{sup 3}H]CGP 61594 labeled with high affinity (K{sub D}=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [{sup 3}H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [{sup 3}H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [{sup 3}H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Focal status epilepticus and progressive dyskinesia: A novel phenotype for glycine receptor antibody-mediated neurological disease in children.

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Chan, D W S; Thomas, T; Lim, M; Ling, S; Woodhall, M; Vincent, A

2017-03-01

Antibody-associated disorders of the central nervous system are increasingly recognised in adults and children. Some are known to be paraneoplastic, whereas in others an infective trigger is postulated. They include disorders associated with antibodies to N-methyl-d-aspartate receptor (NMDAR), voltage-gated potassium channel-complexes (VGKC-complex), GABA B receptor or glycine receptor (GlyR). With antibodies to NMDAR or VGKC-complexes, distinct clinical patterns are well characterised, but as more antibodies are discovered, the spectra of associated disorders are evolving. GlyR antibodies have been detected in patients with progressive encephalopathy with rigidity and myoclonus (PERM), or stiff man syndrome, both rare but disabling conditions. We report a case of a young child with focal seizures and progressive dyskinesia in whom GlyR antibodies were detected. Anticonvulsants and immunotherapy were effective in treating both the seizures and movement disorder with good neurological outcome and with a decline in the patient's serum GlyR-Ab titres. Glycine receptor antibodies are associated with focal status epilepticus and seizures, encephalopathy and progressive dyskinesia and should be evaluated in autoimmune encephalitis. Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Mechanisms of glycine release, which build up synaptic and extrasynaptic glycine levels: the role of synaptic and non-synaptic glycine transporters.

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Harsing, Laszlo G; Matyus, Peter

2013-04-01

Glycine is an amino acid neurotransmitter that is involved in both inhibitory and excitatory neurochemical transmission in the central nervous system. The role of glycine in excitatory neurotransmission is related to its coagonist action at glutamatergic N-methyl-D-aspartate receptors. The glycine levels in the synaptic cleft rise many times higher during synaptic activation assuring that glycine spills over into the extrasynaptic space. Another possible origin of extrasynaptic glycine is the efflux of glycine occurring from astrocytes associated with glutamatergic synapses. The release of glycine from neuronal or glial origins exhibits several differences compared to that of biogenic amines or other amino acid neurotransmitters. These differences appear in an external Ca(2+)- and temperature-dependent manner, conferring unique characteristics on glycine as a neurotransmitter. Glycine transporter type-1 at synapses may exhibit neural and glial forms and plays a role in controlling synaptic glycine levels and the spill over rate of glycine from the synaptic cleft into the extrasynaptic biophase. Non-synaptic glycine transporter type-1 regulates extrasynaptic glycine concentrations, either increasing or decreasing them depending on the reverse or normal mode operation of the carrier molecule. While we can, at best, only estimate synaptic glycine levels at rest and during synaptic activation, glycine concentrations are readily measurable via brain microdialysis technique applied in the extrasynaptic space. The non-synaptic N-methyl-D-aspartate receptor may obtain glycine for activation following its spill over from highly active synapses or from its release mediated by the reverse operation of non-synaptic glycine transporter-1. The sensitivity of non-synaptic N-methyl-D-aspartate receptors to glutamate and glycine is many times higher than that of synaptic N-methyl-D-aspartate receptors making the former type of receptor the primary target for drug action. Synaptic

Early history of glycine receptor biology in mammalian spinal cord circuits

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Robert J Callister

2010-05-01

Full Text Available In this review we provide an overview of key in vivo experiments, undertaken in the cat spinal cord in the 1950s and 1960s, and point out their contributions to our present understanding of glycine receptor (GlyR function. Importantly, some of these discoveries were made well before an inhibitory receptor, or its agonist, was identified. These contributions include the universal acceptance of a chemical mode of synaptic transmission, that GlyRs are chloride channels, are involved in reciprocal and recurrent spinal inhibition, are selectively blocked by strychnine, and can be distinguished from the GABAA receptor by their insensitivity to bicuculline. The early in vivo work on inhibitory mechanisms in spinal neurons also contributed to several enduring principles on synaptic function, such as the time associated with synaptic delay, the extension of Dale’s hypothesis (regarding the chemical unity of nerve cells and their terminals to neurons within the central nervous system, and the importance of inhibition for synaptic integration in motor and sensory circuits. We hope the work presented here will encourage those interested in GlyR biology and inhibitory mechanisms to seek out and read some of the “classic” articles that document the above discoveries.

Glycine receptors in CNS neurons as a target for nonretrograde action of cannabinoids

NARCIS (Netherlands)

Lozovaya, N.; Yatsenko, N.; Beketov, A.; Tsintsadze, T.; Burnashev, N.

2005-01-01

At many central synapses, endocannabinoids released by postsynaptic cells act retrogradely on presynaptic G-protein-coupled cannabinoid receptors to inhibit neurotransmitter release. Here, we demonstrate that cannabinoids may directly affect the functioning of inhibitory glycine receptor (GlyR)

Oxime Ethers of (E)-11-Isonitrosostrychnine as Highly Potent Glycine Receptor Antagonists

DEFF Research Database (Denmark)

Mohsen, Amal M Y; Mandour, Yasmine M; Sarukhanyan, Edita

2016-01-01

of the crystal structure of the α3 glycine receptor indicated the same orientation of the strychnine core for all analogues. For the most potent oxime ethers, the ether substituent was accommodated in a lipophilic receptor binding pocket. The findings identify the oxime hydroxy group as a suitable attachment...

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

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Schaefer, Natascha; Kluck, Christoph J; Price, Kerry L; Meiselbach, Heike; Vornberger, Nadine; Schwarzinger, Stephan; Hartmann, Stephanie; Langlhofer, Georg; Schulz, Solveig; Schlegel, Nadja; Brockmann, Knut; Lynch, Bryan; Becker, Cord-Michael; Lummis, Sarah C R; Villmann, Carmen

2015-01-07

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding. Copyright © 2015 the authors 0270-6474/15/350422-16$15.00/0.

Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

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Orth Angela

2009-04-01

Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

DEFF Research Database (Denmark)

Pless, Stephan Alexander; Lynch, Joseph W

2009-01-01

Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the alpha1 glycine receptor increases during ...

Activation-Dependent Rapid Postsynaptic Clustering of Glycine Receptors in Mature Spinal Cord Neurons

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Eto, Kei; Murakoshi, Hideji; Watanabe, Miho; Hirata, Hiromi; Moorhouse, Andrew J.; Ishibashi, Hitoshi

2017-01-01

Abstract Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal development in vivo. While presynaptic mechanisms of the shift of inhibitory neurotransmission are well investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the first postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in a depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of GlyRs in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for 1 h in the mature stage. Furthermore, live cell-imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca2+ nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission but also the inhibitory synaptic plasticity in mature nervous system. PMID:28197549

Molecular basis of the alternative recruitment of GABA(A) versus glycine receptors through gephyrin

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Maric, Hans-Michael; Kasaragod, Vikram Babu; Hausrat, Torben Johann

2014-01-01

γ-Aminobutyric acid type A and glycine receptors (GABA(A)Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA(A)Rs are important drug targets regulated by direct interactions with the scaffolding protein ge...

Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

DEFF Research Database (Denmark)

Jensen, Anders A.

2005-01-01

The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two rec...

Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site

DEFF Research Database (Denmark)

Kvist, Trine; Steffensen, Thomas Bielefeldt; Greenwood, Jeremy R

2013-01-01

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glyci...... screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design....

Subregion-specific modulation of excitatory input and dopaminergic output in the striatum by tonically activated glycine and GABAA receptors

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Louise eAdermark

2011-10-01

Full Text Available The flow of cortical information through the basal ganglia is a complex spatiotemporal pattern of increased and decreased firing. The striatum is the biggest input nucleus to the basal ganglia and the aim of this study was to assess the role of inhibitory GABAA and glycine receptors in regulating synaptic activity in the dorsolateral (DLS and ventral striatum (nucleus accumbens, nAc. Local field potential recordings from coronal brain slices of juvenile and adult Wistar rats showed that GABAA receptors and strychnine-sensitive glycine receptors are tonically activated and inhibit excitatory input to the DLS and to the nAc. Strychnine-induced disinhibition of glutamatergic transmission was insensitive to the muscarinic receptor inhibitor scopolamine (10 µM, inhibited by the nicotinic acetylcholine receptor antagonist mecamylamine (10 µM and blocked by GABAA receptor inhibitors, suggesting that tonically activated glycine receptors depress excitatory input to the striatum through modulation of cholinergic and GABAergic neurotransmission. As an end-product example of striatal GABAergic output in vivo we measured dopamine release in the DLS and nAc by microdialysis in the awake and freely moving rat. Reversed dialysis of bicuculline (50 μM in perfusate only increased extrasynaptic dopamine levels in the nAc, while strychnine administered locally (200 μM in perfusate decreased dopamine output by 60% in both the DLS and nAc. Our data suggest that GABAA and glycine receptors are tonically activated and modulate striatal transmission in a partially sub-region specific manner.

Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy

DEFF Research Database (Denmark)

Pless, Stephan Alexander; Lynch, Joseph W

2009-01-01

associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage...

Glycine Receptor Activation Impairs ATP-Induced Calcium Transients in Cultured Cortical Astrocytes

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Tatiana P. Morais

2018-01-01

Full Text Available In central nervous system, glycine receptor (GlyR is mostly expressed in the spinal cord and brainstem, but glycinergic transmission related elements have also been identified in the brain. Astrocytes are active elements at the tripartite synapse, being responsible for the maintenance of brain homeostasis and for the fine-tuning of synaptic activity. These cells communicate, spontaneously or in response to a stimulus, by elevations in their cytosolic calcium (calcium transients, Ca2+T that can be propagated to other cells. How these Ca2+T are negatively modulated is yet poorly understood. In this work, we evaluated GlyR expression and its role on calcium signaling modulation in rat brain astrocytes. We first proved that GlyR, predominantly subunits α2 and β, was expressed in brain astrocytes and its localization was confirmed in the cytoplasm and astrocytic processes by immunohistochemistry assays. Calcium imaging experiments in cultured astrocytes showed that glycine (500 μM, a GlyR agonist, caused a concentration-dependent reduction in ATP-induced Ca2+T, an effect abolished by the GlyR antagonist, strychnine (0.8 μM, as well as by nocodazole (1 μM, known to impair GlyR anchorage to the plasma membrane. This effect was mimicked by activation of GABAAR, another Cl--permeable channel. In summary, we demonstrated that GlyR activation in astrocytes mediates an inhibitory effect upon ATP induced Ca2+T, which most probably involves changes in membrane permeability to Cl- and requires GlyR anchorage at the plasma membrane. GlyR in astrocytes may thus be part of a mechanism to modulate astrocyte-to-neuron communication.

Glycine transporter 1 is a target for the treatment of epilepsy

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Shen, Hai-Ying; van Vliet, Erwin A.; Bright, Kerry-Ann; Hanthorn, Marissa; Lytle, Nikki K.; Gorter, Jan; Aronica, Eleonora; Boison, Detlev

2015-01-01

Glycine is the major inhibitory neurotransmitter in brainstem and spinal cord, whereas in hippocampus glycine exerts dual modulatory roles on strychnine-sensitive glycine receptors and on the strychnine-insensitive glycineB site of the N-methyl-D-aspartate receptor (NMDAR). In hippocampus, the

Glycine transporter 1 is a target for the treatment of epilepsy

NARCIS (Netherlands)

Shen, H-Y; van Vliet, E.A.; Bright, K-A.; Hanthorn, M.; Lytle, N.K.; Gorter, J.; Aronica, E.; Boison, D.

2015-01-01

Glycine is the major inhibitory neurotransmitter in brainstem and spinal cord, whereas in hippocampus glycine exerts dual modulatory roles on strychnine-sensitive glycine receptors and on the strychnine-insensitive glycineB site of the N-methyl-d-aspartate receptor (NMDAR). In hippocampus, the

Transient receptor potential channels in essential hypertension

DEFF Research Database (Denmark)

Liu, Daoyan; Scholze, Alexandra; Zhu, Zhiming

2006-01-01

The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated.......The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated....

Identification of AICP as a GluN2C-Selective N-Methyl-d-Aspartate Receptor Superagonist at the GluN1 Glycine Site

DEFF Research Database (Denmark)

Jessen, Maja; Frederiksen, Kristen; Yi, Feng

2017-01-01

N-methyl-d-aspartate (NMDA)-type ionotropic glutamate receptors mediate excitatory neurotransmission in the central nervous system and are critically involved in brain function. NMDA receptors are also implicated in psychiatric and neurological disorders and have received considerable attention....../2A-D), in which DCS is a superagonist at GluN2C-containing receptors compared with glycine and a partial agonist at GluN2B-containing receptors. Here, we identify (R)-2-amino-3-(4-(2-ethylphenyl)-1H-indole-2-carboxamido)propanoic acid (AICP) as a glycine site agonist with unique GluN2-dependent...

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

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Thorsten Pauly

Full Text Available Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs. Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

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Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-07-16

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Alterations in brain extracellular dopamine and glycine levels following combined administration of the glycine transporter type-1 inhibitor Org-24461 and risperidone.

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Nagy, Katalin; Marko, Bernadett; Zsilla, Gabriella; Matyus, Peter; Pallagi, Katalin; Szabo, Geza; Juranyi, Zsolt; Barkoczy, Jozsef; Levay, Gyorgy; Harsing, Laszlo G

2010-12-01

The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D(2) dopamine receptors. N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D(2) dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced

NMDA receptor glycine modulatory site in the ventral tegmental area regulates the acquisition, retrieval, and reconsolidation of cocaine reward memory.

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Zhou, Shuang-jiang; Xue, Li-fen; Wang, Xue-yi; Jiang, Wen-gao; Xue, Yan-xue; Liu, Jian-feng; He, Yin-yin; Luo, Yi-xiao; Lu, Lin

2012-05-01

Accumulating clinical and preclinical studies have shown that the memories of the rewarding effects of drugs and their paired cues may contribute to relapse and persistent cocaine use. Glutaminergic actions in the ventral tegmental area (VTA) have been shown to regulate the rewarding effect of drugs and conditioned responses to drug-associated cues, but the role of the VTA in the acquisition, retrieval, and reconsolidation of cocaine cues is not yet known. In the present study, we used 7-chlorothiokynurenic acid (7-CTKA), an N-methyl-D-aspartate (NMDA) receptor glycine modulatory site antagonist with no rewarding effects, to examine the role of the NMDA receptor glycine modulatory site in the acquisition, retrieval, and reconsolidation of cocaine-related reward memory using the conditioned place preference (CPP) paradigm. Separate groups of Sprague-Dawley rats were trained to acquire cocaine-induced CPP. Vehicle or 7-CTKA was microinjected into the VTA or substantia nigra (SN) (5 μg/μl) at different time points: 10 min before each CPP training session (acquisition), 10 min before the reactivation of CPP (retrieval), and immediately after the reactivation of CPP (reconsolidation). Cocaine-induced CPP was retested 24 h and 1 and 2 weeks after 7-CTKA administration. 7-CTKA microinjected into the VTA, but not SN, significantly impaired the acquisition, retrieval, and reconsolidation of cocaine-induced CPP without affecting cocaine-induced locomotion. Our findings suggest that the NMDA receptor glycine modulatory site in the VTA plays a major role in cocaine reward memory, and NMDA receptor glycine site antagonists may be potential pharmacotherapies for the management of relapse.

Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

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Jensen, Anders A.; Kristiansen, Uffe

2004-01-01

In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput scr...... not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.......In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput...... ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited alpha1 GlyR function, the potentiation of alpha1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may...

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

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Claire N J Meunier

Full Text Available N-methyl-D-aspartate receptors (NMDARs play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

Increased NMDA receptor inhibition at an increased Sevoflurane MAC

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Brosnan Robert J

2012-06-01

Full Text Available Abstract Background Sevoflurane potently enhances glycine receptor currents and more modestly decreases NMDA receptor currents, each of which may contribute to immobility. This modest NMDA receptor antagonism by sevoflurane at a minimum alveolar concentration (MAC could be reciprocally related to large potentiation of other inhibitory ion channels. If so, then reduced glycine receptor potency should increase NMDA receptor antagonism by sevoflurane at MAC. Methods Indwelling lumbar subarachnoid catheters were surgically placed in 14 anesthetized rats. Rats were anesthetized with sevoflurane the next day, and a pre-infusion sevoflurane MAC was measured in duplicate using a tail clamp method. Artificial CSF (aCSF containing either 0 or 4 mg/mL strychnine was then infused intrathecally at 4 μL/min, and the post-infusion baseline sevoflurane MAC was measured. Finally, aCSF containing strychnine (either 0 or 4 mg/mL plus 0.4 mg/mL dizocilpine (MK-801 was administered intrathecally at 4 μL/min, and the post-dizocilpine sevoflurane MAC was measured. Results Pre-infusion sevoflurane MAC was 2.26%. Intrathecal aCSF alone did not affect MAC, but intrathecal strychnine significantly increased sevoflurane requirement. Addition of dizocilpine significantly decreased MAC in all rats, but this decrease was two times larger in rats without intrathecal strychnine compared to rats with intrathecal strychnine, a statistically significant (P  Conclusions Glycine receptor antagonism increases NMDA receptor antagonism by sevoflurane at MAC. The magnitude of anesthetic effects on a given ion channel may therefore depend on the magnitude of its effects on other receptors that modulate neuronal excitability.

An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

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Lynagh, Timothy Peter; Lynch, Joseph W

2010-01-01

The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

One-step preparation of [2,3-{sup 3}H]1-aminocyclo-propanecarboxylic acid: a useful ligand for strychnine-insensitive glycine receptors

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Lewin, A.H.; Lamb, P.B. [Research Triangle Inst., Research Triangle Park, NC (United States); Popik, P.; Skolnick, P. [National Insts. of Health, Bethesda, MD (United States)

1994-11-01

Catalytic hydrogenation of 1-aminocyclopropenecarboxylic acid under tritium gas afforded [2,3-{sup 3}H]1-aminocyclopropanecarboxylic acid with specific activity 26 Ci/mmol, determined by a combination of {sup 1}H and {sup 3}H NMR. Pilot radioligand binding assays indicate this compound will be a useful prove for the NMDA receptor-associated strychnine-insensitive glycine receptor. (author).

Transient receptor potential channel superfamily: Role in lower urinary tract function.

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Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

2015-11-01

Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

A Structural and Mutagenic Blueprint for Molecular Recognition of Strychnine and d-Tubocurarine by Different Cys-Loop Receptors

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Brams, M.; Pandya, A.; Kuzmin, D.; van Elk, R.; Krijnen, L.; Yakel, J.L.; Tsetlin, V.; Smit, A.B.; Ulens, C.

2011-01-01

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic

Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

International Nuclear Information System (INIS)

Gillberg, P.-G.; Aquilonius, S.-M.

1985-01-01

Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

Principles of agonist recognition in Cys-loop receptors

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Timothy eLynagh

2014-04-01

Full Text Available Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine and GABA. After the term chemoreceptor emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands.

Sigma-1 Receptor Plays a Negative Modulation on N-type Calcium Channel

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Kang Zhang

2017-05-01

Full Text Available The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca2+ channel is still unclear. Considering both sigma-1 receptors and N-type Ca2+ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR and immunofluorescence staining. N-type Ca2+ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084 were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063. Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca2+ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca2+ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET assays and co-immunoprecipitation (Co-IP demonstrated that sigma-1 receptors and N-type Ca2+ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca2+ channels. The mechanism for the inhibition of sigma-1 receptors on

Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions.

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Musante, Veronica; Summa, Maria; Cunha, Rodrigo A; Raiteri, Maurizio; Pittaluga, Anna

2011-05-01

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Sigma-1 receptor agonists directly inhibit Nav1.2/1.4 channels.

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Xiao-Fei Gao

Full Text Available (+-SKF 10047 (N-allyl-normetazocine is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+-SKF 10047 inhibits K(+, Na(+ and Ca2+ channels via sigma-1 receptor activation. We found that (+-SKF 10047 inhibited Na(V1.2 and Na(V1.4 channels independently of sigma-1 receptor activation. (+-SKF 10047 equally inhibited Na(V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+-SKF 10047 inhibition of Na(V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM and 1,3-di-o-tolyl-guanidine (DTG also inhibited Na(V1.2 currents through a sigma-1 receptor-independent pathway. The (+-SKF 10047 inhibition of Na(V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe(1764 and Tyr(1771 in the IV-segment 6 domain of the Na(V1.2 channel and Phe(1579 in the Na(V1.4 channel for (+-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na(V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.

The Role of Canonical Transient Receptor Potential Channels in Seizure and Excitotoxicity

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Fang Zheng

2014-04-01

Full Text Available Canonical transient receptor potential (TRPC channels are a family of polymodal cation channels with some degree of Ca2+ permeability. Although initially thought to be channels mediating store-operated Ca2+ influx, TRPC channels can be activated by stimulation of Gq-coupled G-protein coupled receptors, or by an increase in intracellular free Ca2+ concentration. Thus, activation of TRPC channels could be a common downstream event of many signaling pathways that contribute to seizure and excitotoxicity, such as N-methyl-D-aspartate (NMDA receptor-mediated Ca2+ influx, or metabotropic glutamate receptor activation. Recent studies with genetic ablation of various TRPC family members have demonstrated that TRPC channels, in particular heteromeric TRPC1/4 channels and homomeric TRPC5 channels, play a critical role in both pilocarpine-induced acute seizures and neuronal cell death. However, exact underlying mechanisms remain to be fully elucidated, and selective TRPC modulators and antibodies with better specificity are urgently needed for future research.

A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

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Kostenis, Evi; Martini, Lene; Ellis, James

2004-01-01

Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

GABA and glycine as neurotransmitters: a brief history.

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Bowery, N G; Smart, T G

2006-01-01

gamma-Aminobutyric acid (GABA) emerged as a potentially important brain chemical just over 50 years ago, but its significance as a neurotransmitter was not fully realized until over 16 years later. We now know that at least 40% of inhibitory synaptic processing in the mammalian brain uses GABA. Establishing its role as a transmitter was a lengthy process and it seems hard to believe with our current knowledge that there was ever any dispute about its role in the mammalian brain. The detailed information that we now have about the receptors for GABA together with the wealth of agents which facilitate or reduce GABA receptor mechanisms make the prospects for further research very exciting. The emergence of glycine as a transmitter seems relatively painless by comparison to GABA. Perhaps this is appropriate for the simplest of transmitter structures! Its discovery within the spinal cord and brainstem approximately 40 years ago was followed only 2 years later by the proposal that it be conferred with 'neurotransmitter' status. It was another 16 years before the receptor was biochemically isolated. Now it is readily accepted as a vital spinal and supraspinal inhibitory transmitter and we know many details regarding its molecular structure and trafficking around neurones. The pharmacology of these receptors has lagged behind that of GABA. There is not the rich variety of allosteric modulators that we have come to readily associate with GABA receptors and which has provided us with a virtual treasure trove of important drugs used in anxiety, insomnia, epilepsy, anaesthesia, and spasticity, all stemming from the actions of the simple neutral amino acid GABA. Nevertheless, the realization that glycine receptors are involved in motor reflexes and nociceptive pathways together with the more recent advent of drugs that exhibit some subtype selectivity make the goal of designing selective therapeutic ligands for the glycine receptor that much closer.

Glycine receptors in the human substantia nigra as defined by (3H)strychnine binding

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de Montis, G; Beaumont, K; Javoy-Agid, F; Agid, Y; Constandinidis, J; Lowenthal, A; Lloyd, K G

1982-03-01

Specific (3H)strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity KD (3--8 nM) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 nM, respectively) and rat substantia nigra (12 nM). In the human brain the distribution of (3H)strychnine binding (at 10 nM) was: spinal cord . substantia nigra, pars compacta greater than substantia nigra, pars reticulata . inferior olivary nucleus greater than cerebral cortex. The binding capacity (Bmax) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. (3H)Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, (3H)strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. (3H)Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.

High throughput techniques for discovering new glycine receptor modulators and their binding sites

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Daniel F Gilbert

2009-10-01

Full Text Available The inhibitory glycine receptor (GlyR is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific compounds. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384 well plate using nanogram quantities of cDNA.

The Prodrug 4-Chlorokynurenine Causes Ketamine-Like Antidepressant Effects, but Not Side Effects, by NMDA/GlycineB-Site Inhibition.

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Zanos, Panos; Piantadosi, Sean C; Wu, Hui-Qiu; Pribut, Heather J; Dell, Matthew J; Can, Adem; Snodgrass, H Ralph; Zarate, Carlos A; Schwarcz, Robert; Gould, Todd D

2015-10-01

Currently approved antidepressant drug treatment typically takes several weeks to be effective. The noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine has shown efficacy as a rapid-acting treatment of depression, but its use is associated with significant side effects. We assessed effects following blockade of the glycineB co-agonist site of the NMDA receptor, located on the GluN1 subunit, by the selective full antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), delivered by systemic administration of its brain-penetrant prodrug 4-chlorokynurenine (4-Cl-KYN) in mice. Following administration of 4-Cl-KYN, 7-Cl-KYNA was promptly recovered extracellularly in hippocampal microdialysate of freely moving animals. The behavioral responses of the animals were assessed using measures of ketamine-sensitive antidepressant efficacy (including the 24-hour forced swim test, learned helplessness test, and novelty-suppressed feeding test). In these tests, distinct from fluoxetine, and similar to ketamine, 4-Cl-KYN administration resulted in rapid, dose-dependent and persistent antidepressant-like effects following a single treatment. The antidepressant effects of 4-Cl-KYN were prevented by pretreatment with glycine or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). 4-Cl-KYN administration was not associated with the rewarding and psychotomimetic effects of ketamine, and did not induce locomotor sensitization or stereotypic behaviors. Our results provide further support for antagonism of the glycineB site for the rapid treatment of treatment-resistant depression without the negative side effects seen with ketamine or other channel-blocking NMDA receptor antagonists. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Opportunistic activation of TRP receptors by endogenous lipids: exploiting lipidomics to understand TRP receptor cellular communication.

Science.gov (United States)

Bradshaw, Heather B; Raboune, Siham; Hollis, Jennifer L

2013-03-19

Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining "orphans." That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are "promiscuous" in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically "opportunistic" in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an "orphan" lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine-binding site of N-methyl-D-aspartate receptor.

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Moriguchi, Shigeki; Tanaka, Tomoya; Narahashi, Toshio; Fukunaga, Kohji

2013-10-01

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

International Nuclear Information System (INIS)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-01

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

Science.gov (United States)

Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

2016-01-01

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

Discovery of a novel allosteric modulator of 5-HT3 receptor

DEFF Research Database (Denmark)

Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B

2012-01-01

The ligand-gated ion channels in the Cysloop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA and glycine. Cysloop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel...... receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)subunit and TM1 and TM2 in the (minus)subunit. The Ser248, Leu288, Ile290, Thr294 and Gly306 residues are identified as important...

Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

DEFF Research Database (Denmark)

Pless, Stephan Alexander; Lynch, Joseph W

2009-01-01

, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E....... At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes...... in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded...

Functional Annotation of Ion Channel Structures by Molecular Simulation.

Science.gov (United States)

Trick, Jemma L; Chelvaniththilan, Sivapalan; Klesse, Gianni; Aryal, Prafulla; Wallace, E Jayne; Tucker, Stephen J; Sansom, Mark S P

2016-12-06

Ion channels play key roles in cell membranes, and recent advances are yielding an increasing number of structures. However, their functional relevance is often unclear and better tools are required for their functional annotation. In sub-nanometer pores such as ion channels, hydrophobic gating has been shown to promote dewetting to produce a functionally closed (i.e., non-conductive) state. Using the serotonin receptor (5-HT 3 R) structure as an example, we demonstrate the use of molecular dynamics to aid the functional annotation of channel structures via simulation of the behavior of water within the pore. Three increasingly complex simulation analyses are described: water equilibrium densities; single-ion free-energy profiles; and computational electrophysiology. All three approaches correctly predict the 5-HT 3 R crystal structure to represent a functionally closed (i.e., non-conductive) state. We also illustrate the application of water equilibrium density simulations to annotate different conformational states of a glycine receptor. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of sarcosine and N, N-dimethylglycine on NMDA receptor-mediated excitatory field potentials.

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Lee, Mei-Yi; Lin, Yi-Ruu; Tu, Yi-Shu; Tseng, Yufeng Jane; Chan, Ming-Huan; Chen, Hwei-Hsien

2017-02-28

Sarcosine, a glycine transporter type 1 inhibitor and an N-methyl-D-aspartate (NMDA) receptor co-agonist at the glycine binding site, potentiates NMDA receptor function. Structurally similar to sarcosine, N,N-dimethylglycine (DMG) is also N-methyl glycine-derivative amino acid and commonly used as a dietary supplement. The present study compared the effects of sarcosine and DMG on NMDA receptor-mediated excitatory field potentials (EFPs) in mouse medial prefrontal cortex brain slices using a multi-electrode array system. Glycine, sarcosine and DMG alone did not alter the NMDA receptor-mediated EFPs, but in combination with glutamate, glycine and its N-methyl derivatives significantly increased the frequency and amplitude of EFPs. The enhancing effects of glycine analogs in combination with glutamate on EFPs were remarkably reduced by the glycine binding site antagonist 7-chlorokynurenate (7-CK). However, DMG, but not sarcosine, reduced the frequency and amplitude of EFPs elicited by co-application of glutamate plus glycine. D-cycloserine, a partial agonist at the glycine binding site on NMDA receptors, affected EFPs in a similar manner to DMG. Furthermore, DMG, but not sarcosine, reduced the frequencies and amplitudes of EFPs elicited by glutamate plus D-serine, another endogenous ligand for glycine binding site. These findings suggest that sarcosine acts as a full agonist, yet DMG is a partial agonist at glycine binding site of NMDA receptors. The molecular docking analysis indicated that the interactions of glycine, sarcosine, and DMG to NMDA receptors are highly similar, supporting that the glycine binding site of NMDA receptors is a critical target site for sarcosine and DMG.

Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

Science.gov (United States)

Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

2010-07-15

In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors.

Science.gov (United States)

Guo, Xiaoli; Chronis, Demosthenis; De La Torre, Carola M; Smeda, John; Wang, Xiaohong; Mitchum, Melissa G

2015-08-01

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant-parasitic cyst nematodes secrete CLE-like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode-induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock-down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Influence of high glycine diets on the activity of glycine-catabolizing enzymes and on glycine catabolism in rats

International Nuclear Information System (INIS)

Petzke, K.J.; Albrecht, V.; Przybilski, H.

1986-01-01

Male albino rats were adapted to isocaloric purified diets that differed mainly in their glycine and casein contents. Controls received a 30% casein diet. In experimental diets gelatin or gelatin hydrolysate was substituted for half of the 30% casein. An additional group was fed a glycine-supplemented diet, which corresponded in glycine level to the gelatin diet but in which the protein level was nearly the same as that of the casein control diet. Another group received a 15% casein diet. Rat liver glycine cleavage system, serine hydroxymethyltransferase and serine dehydratase activities were measured. 14 CO 2 production from the catabolism of 14 C-labeled glycine was measured in vivo and in vitro (from isolated hepatocytes). Serine dehydratase and glycine cleavage system activities were higher in animals fed 30% casein diets than in those fed 15% casein diets. Serine hydroxymethyltransferase activity of the cytosolic and mitochondrial fractions was highest when a high glycine diet (glycine administered as pure, protein bound in gelatin or peptide bound in gelatin hydrolysate) was fed. 14 CO 2 formation from [1- 14 C]- and [2- 14 C]glycine both in vivo and in isolated hepatocytes was higher when a high glycine diet was fed than when a casein diet was fed. These results suggest that glycine catabolism is dependent on and adaptable to the glycine content of the diet. Serine hydroxymethyltransferase appears to play a major role in the regulation of glycine degradation via serine and pyruvate

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors

DEFF Research Database (Denmark)

Kristensen, Anders S; Hansen, Kasper B; Naur, Peter

2016-01-01

-term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify...

Effects of a glycine transporter-1 inhibitor and D-serine on MK-801-induced immobility in the forced swimming test in rats.

Science.gov (United States)

Kawaura, Kazuaki; Koike, Hiroyuki; Kinoshita, Kohnosuke; Kambe, Daiji; Kaku, Ayaka; Karasawa, Jun-ichi; Chaki, Shigeyuki; Hikichi, Hirohiko

2015-02-01

Glutamatergic dysfunction, particularly the hypofunction of N-methyl-D-aspartate (NMDA) receptors, is involved in the pathophysiology of schizophrenia. The positive modulation of the glycine site on the NMDA receptor has been proposed as a novel therapeutic approach for schizophrenia. However, its efficacy against negative symptoms, which are poorly managed by current medications, has not been fully addressed. In the present study, the effects of the positive modulation of the glycine site on the NMDA receptor were investigated in an animal model of negative symptoms of schizophrenia. The subchronic administration of MK-801 increased immobility in the forced swimming test in rats without affecting spontaneous locomotor activity. The increased immobility induced by MK-801 was attenuated by the atypical antipsychotic clozapine but not by either the typical antipsychotic haloperidol or the antidepressant imipramine, indicating that the increased immobility induced by subchronic treatment with MK-801 in the forced swimming test may represent a negative symptom of schizophrenia. Likewise, positive modulation of the glycine sites on the NMDA receptor using an agonist for the glycine site, D-serine, and a glycine transporter-1 inhibitor, N-[(3R)-3-([1,1'-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydrochloride (NFPS), significantly reversed the increase in immobility in MK-801-treated rats without reducing the immobility time in vehicle-treated rats. The present results show that the stimulation of the NMDA receptor through the glycine site on the receptor either directly with D-serine or by blocking glycine transporter-1 attenuates the immobility elicited by the subchronic administration of MK-801 and may be potentially useful for the treatment of negative symptoms of schizophrenia. Copyright © 2014 Elsevier B.V. All rights reserved.

Double-Nanodomain Coupling of Calcium Channels, Ryanodine Receptors, and BK Channels Controls the Generation of Burst Firing.

Science.gov (United States)

Irie, Tomohiko; Trussell, Laurence O

2017-11-15

Action potentials clustered into high-frequency bursts play distinct roles in neural computations. However, little is known about ionic currents that control the duration and probability of these bursts. We found that, in cartwheel inhibitory interneurons of the dorsal cochlear nucleus, the likelihood of bursts and the interval between their spikelets were controlled by Ca 2+ acting across two nanodomains, one between plasma membrane P/Q Ca 2+ channels and endoplasmic reticulum (ER) ryanodine receptors and another between ryanodine receptors and large-conductance, voltage- and Ca 2+ -activated K + (BK) channels. Each spike triggered Ca 2+ -induced Ca 2+ release (CICR) from the ER immediately beneath somatic, but not axonal or dendritic, plasma membrane. Moreover, immunolabeling demonstrated close apposition of ryanodine receptors and BK channels. Double-nanodomain coupling between somatic plasma membrane and hypolemmal ER cisterns provides a unique mechanism for rapid control of action potentials on the millisecond timescale. Copyright © 2017 Elsevier Inc. All rights reserved.

The purinergic P2X7 ion channel receptor — a ‘repair’ receptor in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye

2018-01-01

A strong skeleton relies on adaptation to varying physical demands and on maintenance of the bone tissue in order to avoid accumulation of micro-damage. In bone, the purinergic P2X7 ion channel receptor is expressed on both cells of the stromal lineage such as the bone forming osteoblasts...... and the mechano-sensing osteocytes and on cells belonging to the immune-related monocyte–macrophage lineage, the bone resorbing osteoclasts. Recent studies have demonstrated that the receptor plays important roles in the anabolic responses to mechanical loading on bone and, together with the pannexin1 hemi......-channel, in the process of initiating bone remodeling in response to micro-damage. Thus, the receptor is crucial in skeletal mechano-transduction and in the continuous repair process. However, under pathophysiological conditions such as diabetes with high glucose concentrations or glucocorticoid-treatment the receptor...

High Concentrations of Tranexamic Acid Inhibit Ionotropic Glutamate Receptors.

Science.gov (United States)

Lecker, Irene; Wang, Dian-Shi; Kaneshwaran, Kirusanthy; Mazer, C David; Orser, Beverley A

2017-07-01

The antifibrinolytic drug tranexamic acid is structurally similar to the amino acid glycine and may cause seizures and myoclonus by acting as a competitive antagonist of glycine receptors. Glycine is an obligatory co-agonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Thus, it is plausible that tranexamic acid inhibits NMDA receptors by acting as a competitive antagonist at the glycine binding site. The aim of this study was to determine whether tranexamic acid inhibits NMDA receptors, as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate subtypes of ionotropic glutamate receptors. Tranexamic acid modulation of NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate receptors was studied using whole cell voltage-clamp recordings of current from cultured mouse hippocampal neurons. Tranexamic acid rapidly and reversibly inhibited NMDA receptors (half maximal inhibitory concentration = 241 ± 45 mM, mean ± SD; 95% CI, 200 to 281; n = 5) and shifted the glycine concentration-response curve for NMDA-evoked current to the right. Tranexamic acid also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (half maximal inhibitory concentration = 231 ± 91 mM; 95% CI, 148 to 314; n = 5 to 6) and kainate receptors (half maximal inhibitory concentration = 90 ± 24 mM; 95% CI, 68 to 112; n = 5). Tranexamic acid inhibits NMDA receptors likely by reducing the binding of the co-agonist glycine and also inhibits α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors. Receptor blockade occurs at high millimolar concentrations of tranexamic acid, similar to the concentrations that occur after topical application to peripheral tissues. Glutamate receptors in tissues including bone, heart, and nerves play various physiologic roles, and tranexamic acid inhibition of these receptors may contribute to adverse drug effects.

Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

Science.gov (United States)

Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

2018-04-01

Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.

Directory of Open Access Journals (Sweden)

Sahil Talwar

Full Text Available Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems

Post-translational regulation of P2X receptor channels: modulation by phospholipids

Directory of Open Access Journals (Sweden)

Louis-Philippe eBernier

2013-11-01

Full Text Available P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane.All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e. homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C-linked metabotropic receptors and P2X receptor channels in DRG sensory neurons and microglia.

Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons

Science.gov (United States)

Tong, Gary; Takahashi, Hiroto; Tu, Shichun; Shin, Yeonsook; Talantova, Maria; Zago, Wagner; Xia, Peng; Nie, Zhiguo; Goetz, Thomas; Zhang, Dongxian; Lipton, Stuart A.; Nakanishi, Nobuki

2015-01-01

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-D-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extra-synaptic receptors, likely composed of NR1, NR2, and NR3 subunits. PMID:18003876

Autoantibodies to neurotransmitter receptors and ion channels: from neuromuscular to neuropsychiatric disorders

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Pilar eMartinez-Martinez

2013-09-01

Full Text Available Changes of voltage-gated ion channels and ligand-gated receptor channels caused by mutation or autoimmune attack are the cause of so-called channelopathies in the central and peripheral nervous system. We present the pathophysiology of channelopathies of the neuromuscular junction in terms of loss-of-function and gain-of-function principles. Autoantibodies generally have reduced access to the CNS, but in some cases this is enough to cause disease. A review is provided of recent findings implicating autoantibodies against ligand–activated receptor channels and potassium channels in psychiatric and neurological disorders, including schizophrenia and limbic encephalitis. The emergence of channelopathy-related neuropsychiatric disorders has implications for research and practice.

Analgesic effect of GT-0198, a structurally novel glycine transporter 2 inhibitor, in a mouse model of neuropathic pain

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Yu Omori

2015-03-01

Full Text Available This study was conducted to identify the characteristic pharmacological features of GT-0198 that is phenoxymethylbenzamide derivatives. GT-0198 inhibited the function of glycine transporter 2 (GlyT2 in human GlyT2-expressing HEK293 cells and did not bind various major transporters or receptors of neurotransmitters in a competitive manner. Thus, GT-0198 is considered to be a comparatively selective GlyT2 inhibitor. Intravenous, oral, and intrathecal injections of GT-0198 decreased the pain-related response in a model of neuropathic pain with partial sciatic nerve ligation. This result suggests that GT-0198 has an analgesic effect. The analgesic effect of GT-0198 was abolished by the intrathecal injection of strychnine, a glycine receptor antagonist. Therefore, GT-0198 is considered to exhibit its analgesic effect via the activation of a glycine receptor by glycine following presynaptic GlyT2 inhibition in the spinal cord. In summary, GT-0198 is a structurally novel GlyT2 inhibitor bearing a phenoxymethylbenzamide moiety with in vivo efficacy in behavioral models of neuropathic pain.

Novel agents acting on GABA2 receptors: potential cognitive enhancers

International Nuclear Information System (INIS)

Chebib, M.

2001-01-01

γ- Aminobutyric acid (GABA) is a low molecular weight ammo acid found throughout the central and peripheral nervous systems. It is a very flexible molecule and thus can attain a number of low-energy conformations which are recognised by a series of enzymes, receptors and transporter systems. This article will concentrate on the effects of GABA C as the major inhibitory neurotransmitter in the brain. GABA C receptors belong to the superfamily of ligand-gated ion channels that include nicotinic acetylcholine, GABA A , strychnine-sensitive glycine, and serotonin type 3 receptors. The compound outlined in this article provide us with novel leads for the design and development of compounds that may be selective for GABA receptors. Such compounds will help in the study of GABA C receptors both in vitro and in vivo, providing an insight into the role these receptors play in the brain

Ion channels in EEG: isolating channel dysfunction in NMDA receptor antibody encephalitis.

Science.gov (United States)

Symmonds, Mkael; Moran, Catherine H; Leite, M Isabel; Buckley, Camilla; Irani, Sarosh R; Stephan, Klaas Enno; Friston, Karl J; Moran, Rosalyn J

2018-04-30

Neurological and psychiatric practice frequently lack diagnostic probes that can assess mechanisms of neuronal communication non-invasively in humans. In N-methyl-d-aspartate (NMDA) receptor antibody encephalitis, functional molecular assays are particularly important given the presence of NMDA antibodies in healthy populations, the multifarious symptomology and the lack of radiological signs. Recent advances in biophysical modelling techniques suggest that inferring cellular-level properties of neural circuits from macroscopic measures of brain activity is possible. Here, we estimated receptor function from EEG in patients with NMDA receptor antibody encephalitis (n = 29) as well as from encephalopathic and neurological patient controls (n = 36). We show that the autoimmune patients exhibit distinct fronto-parietal network changes from which ion channel estimates can be obtained using a microcircuit model. Specifically, a dynamic causal model of EEG data applied to spontaneous brain responses identifies a selective deficit in signalling at NMDA receptors in patients with NMDA receptor antibody encephalitis but not at other ionotropic receptors. Moreover, though these changes are observed across brain regions, these effects predominate at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons. Given that EEG is a ubiquitously available clinical method, our findings suggest a unique re-purposing of EEG data as an assay of brain network dysfunction at the molecular level.

The role of transient receptor potential channels in joint diseases.

Science.gov (United States)

Krupkova, O; Zvick, J; Wuertz-Kozak, K

2017-10-10

Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

The role of transient receptor potential channels in joint diseases

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O Krupkova

2017-10-01

Full Text Available ransient receptor potential channels (TRP channels are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD cells is largely unexplored. Osteoarthritis (OA and degenerative disc disease (DDD are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

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Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

2016-09-01

Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. Copyright © 2016 Elsevier GmbH. All rights reserved.

P2X receptor channels in endocrine glands

Czech Academy of Sciences Publication Activity Database

Stojilkovic, S. S.; Zemková, Hana

2013-01-01

Roč. 2, č. 4 (2013), s. 173-180 ISSN 2190-460X R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : ATP * purinergic P2X receptor channels * pituitary * endocrine glands Subject RIV: ED - Physiology

Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

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Christine Laura Dixon

2015-12-01

Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

A critical role for glycine transporters in hyperexcitability disorders

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Robert J Harvey

2008-03-01

Full Text Available Defects in mammalian glycinergic neurotransmission result in a complex motor disorder characterized by neonatal hypertonia and an exaggerated startle refl ex, known as hyperekplexia (OMIM 149400. This affects newborn children and is characterized by noise or touch-induced seizures that result in muscle stiffness and breath-holding episodes. Although rare, this disorder can have serious consequences, including brain damage and/or sudden infant death. The primary cause of hyperekplexia is missense and nonsense mutations in the glycine receptor (GlyR α1 subunit gene (GLRA1 on chromosome 5q33.1, although we have also discovered rare mutations in the genes encoding the GlyR β subunit (GLRB and the GlyR clustering proteins gephyrin (GPNH and collybistin (ARHGEF9. Recent studies of the Na+ /Cl--dependent glycine transporters GlyT1 and GlyT2 using mouse knockout models and human genetics have revealed that mutations in GlyT2 are a second major cause of hyperekplexia, while the phenotype of the GlyT1 knockout mouse resembles a devastating neurological disorder known as glycine encephalopathy (OMIM 605899. These findings highlight the importance of these transporters in regulating the levels of synaptic glycine.

Ion channel profile of TRPM8 cold receptors reveals a novel role of TASK-3 potassium channels in thermosensation

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Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L.; Bayliss, Douglas A.; Viana, Félix

2017-01-01

Summary Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold sensitive neurons, combining BAC transgenesis with a molecular profiling approach in FACS purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3 and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a novel role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

Interaction between Ca++-channel antagonists and α2-adrenergic receptors in rabbit ileal cell membrane

International Nuclear Information System (INIS)

Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

1986-01-01

An interaction between Ca ++ -channel antagonists and the α 2 -adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an α 2 -agonist, stimulated NaCl absorption apparently by Ca ++ -channel antagonism since it inhibited 45 Ca ++ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca ++ -channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of 3 H-yohimbine, a specific α 2 -adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd ++ inhibited the specific binding of 3 H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca ++ -channels and α 2 -adrenergic receptors in ileal basolateral membranes. Some Ca ++ -channel antagonists alter α 2 -adrenergic binding to the receptor and α 2 -agonist binding leads to changes in Ca ++ entry. A close spatial relationship between the Ca ++ -channel and the α 2 -receptor could explain the data

Enantiomers of HA-966 (3-amino-1-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects: (+)-HA-966 is a selective glycine/N-methyl-D-aspartate receptor antagonist, but (-)-HA-966 is a potent gamma-butyrolactone-like sedative

International Nuclear Information System (INIS)

Singh, L.; Donald, A.E.; Foster, A.C.; Hutson, P.H.; Iversen, L.L.; Iversen, S.D.; Kemp, J.A.; Leeson, P.D.; Marshall, G.R.; Oles, R.J.; Priestley, T.; Thorn, L.; Tricklebank, M.D.; Vass, C.A.; Williams, B.J.

1990-01-01

The antagonist effect of ±-3-amino-1-hydroxypyrrolid-2-one (HA-966) at the N-methyl-D-aspartate (NMDA) receptor occurs through a selective interaction with the glycine modulatory site within the receptor complex. When the enantiomers of ±-HA-966 were resolved, the (R)-(+)-enantiomer was found to be a selective glycine/NMDA receptor antagonist, a property that accounts for its anticonvulsant activity in vivo. In contrast, the (S)-(-)-enantiomer was only weakly active as an NMDA-receptor antagonist, but nevertheless it possessed a marked sedative and muscle relaxant action in vivo. In radioligand binding experiments, (+)-HA-966 inhibited strychnine-insensitive [ 3 H]glycine binding to rat cerebral cortex synaptic membranes with an IC 50 of 12.5 μM, whereas (-)-HA-966 had an IC 50 value of 339 μM. In mice, (+)-HA-966 antagonized sound and N-methyl-DL-aspartic acid (NMDLA)-induced seizures. The coadministration of D-serine dose-dependently antagonized the anticonvulsant effect of a submaximal dose of (+)-HA-966 against NMDLA-induced seizures. The sedative/ataxic effect of racemic HA-966 was mainly attributable to the (-)-enantiomer. It is suggested that, as in the case of the sedative γ-butyrolactone, disruption of striatal dopaminergic mechanisms may be responsible for this action

Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

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Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

Aboveground feeding by soybean aphid, Aphis glycines, affects soybean cyst nematode, Heterodera glycines, reproduction belowground.

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Michael T McCarville

Full Text Available Heterodera glycines is a cyst nematode that causes significant lost soybean yield in the U.S. Recent studies observed the aphid Aphis glycines and H. glycines interacting via their shared host, soybean, Glycine max. A greenhouse experiment was conducted to discern the effect of A. glycines feeding on H. glycines reproduction. An H. glycines-susceptible cultivar, Kenwood 94, and a resistant cultivar, Dekalb 27-52, were grown in H. glycines-infested soil for 30 and 60 d. Ten days after planting, plants were infested with either zero, five, or ten aphids. At 30 and 60 d, the number of H. glycines females and cysts (dead females and the number of eggs within were counted. In general, H. glycines were less abundant on the resistant than the susceptible cultivar, and H. glycines abundance increased from 30 to 60 d. At 30 d, 33% more H. glycines females and eggs were produced on the resistant cultivar in the ten-aphid treatment compared to the zero-aphid treatment. However, at 30 d the susceptible cultivar had 50% fewer H. glycines females and eggs when infested with ten aphids. At 60 d, numbers of H. glycines females and cysts and numbers of eggs on the resistant cultivar were unaffected by A. glycines feeding, while numbers of both were decreased by A. glycines on the susceptible cultivar. These results indicate that A. glycines feeding improves the quality of soybean as a host for H. glycines, but at higher herbivore population densities, this effect is offset by a decrease in resource quantity.

Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients.

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Andreas Binder

Full Text Available Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K was associated with the presence of paradoxical heat sensation (p = 0.03, and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V with cold hypoalgesia (p = 0.0035. Two main subgroups characterized by preserved (1 and impaired (2 sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and pG (rs222747, M315I to cold hypaesthesia (p = 0.002, but there was absence of associations in subgroup 2. In this study we found no evidence that genetic

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

Science.gov (United States)

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

Ginkgolides and glycine receptors

DEFF Research Database (Denmark)

Jaracz, Stanislav; Nakanishi, Koji; Jensen, Anders A.

2004-01-01

Ginkgolides from the Ginkgo biloba tree are diterpenes with a cage structure consisting of six five-membered rings and a unique tBu group. They exert a variety of biological properties. In addition to being antagonists of the platelet activating factor receptor (PAFR), it has recently been shown ...

The human transient receptor potential vanilloid 3 channel is sensitized via the ERK pathway

Czech Academy of Sciences Publication Activity Database

Vyklická, Lenka; Boukalová, Štěpána; Mačíková, Lucie; Chvojka, Štěpán; Vlachová, Viktorie

2017-01-01

Roč. 292, č. 51 (2017), s. 21083-21091 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA15-15839S Institutional support: RVO:67985823 Keywords : epidermal growth factor receptor (EGFR) * extracellular-signal-regulated kinase (ERK) * keratinocyte * phosphorylation * transient receptor potential channels * TRP channels Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.125, year: 2016

Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation?

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Natascha eSchaefer

2012-10-01

Full Text Available Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400 with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans, single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the α (spasmodic, oscillator, cincinnati, Nmf11 or the β (spastic subunit of the GlyR are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABAA receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator, displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation.

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

Science.gov (United States)

Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

2018-03-16

Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

GLYX-13, an NMDA receptor glycine site functional partial agonist enhances cognition and produces antidepressant effects without the psychotomimetic side effects of NMDA receptor antagonists.

Science.gov (United States)

Moskal, Joseph R; Burch, Ronald; Burgdorf, Jeffrey S; Kroes, Roger A; Stanton, Patric K; Disterhoft, John F; Leander, J David

2014-02-01

The N-methyl-d-aspartate receptor-ionophore complex plays a key role in learning and memory and has efficacy in animals and humans with affective disorders. GLYX-13 is an N-methyl-d-aspartate receptor (NMDAR) glycine-site functional partial agonist and cognitive enhancer that also shows rapid antidepressant activity without psychotomimetic side effects. The authors review the mechanism of action of GLYX-13 that was investigated in preclinical studies and evaluated in clinical studies. Specifically, the authors review its pharmacology, pharmacokinetics, and drug safety that were demonstrated in clinical studies. NMDAR full antagonists can produce rapid antidepressant effects in treatment-resistant subjects; however, they are often accompanied by psychotomimetic effects that make chronic use outside of a clinical trial inpatient setting problematic. GLYX-13 appears to exert its antidepressant effects in the frontal cortex via NMDAR-triggered synaptic plasticity. Understanding the mechanistic underpinning of GLYX-13's antidepressant action should provide both novel insights into the role of the glutamatergic system in depression and identify new targets for therapeutic development.

Channel opening of γ-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

International Nuclear Information System (INIS)

Cash, D.J.; Subbarao, K.

1987-01-01

The function of γ-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36 Cl - isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36 Cl - influx was completed in three phases of ca. 3% (t/sub 1/2/ = 0.6 s), 56% (t/sub 1/2 = 82 s), and 41% (t/sub 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36 Cl - influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles. The half-response concentrations were similar for both receptors. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of low affinity to high-affinity GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria. For both receptors, the measurements over a 2000-fold GABA concentration range required a minimal mechanism involving the occupation of both of the two GABA binding sites for significant channel opening; then the receptors were ca. 80% open. Similarly for both receptors, desensitization was mediated by a different pair of binding sites, although desensitization with only one ligand molecule bound could occur at a 20-fold slower rate

Pharmacological characterization of NMDA-like receptors in the single-celled organism Paramecium primaurelia.

Science.gov (United States)

Ramoino, Paola; Candiani, Simona; Pittaluga, Anna Maria; Usai, Cesare; Gallus, Lorenzo; Ferrando, Sara; Milanese, Marco; Faimali, Marco; Bonanno, Giambattista

2014-02-01

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus

Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

Science.gov (United States)

Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

2011-01-01

We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

Glycine Increases Insulin Sensitivity and Glutathione Biosynthesis and Protects against Oxidative Stress in a Model of Sucrose-Induced Insulin Resistance

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Mohammed El-Hafidi

2018-01-01

Full Text Available Oxidative stress and redox status play a central role in the link between insulin resistance (IR and lipotoxicity in metabolic syndrome. This mechanistic link may involve alterations in the glutathione redox state. We examined the effect of glycine supplementation to diet on glutathione biosynthesis, oxidative stress, IR, and insulin cell signaling in liver from sucrose-fed (SF rats characterized by IR and oxidative stress. Our hypothesis is that the correction of glutathione levels by glycine treatment leads to reduced oxidative stress, a mechanism associated with improved insulin signaling and IR. Glycine treatment decreases the levels of oxidative stress markers in liver from SF rats and increases the concentrations of glutathione (GSH and γ-glutamylcysteine and the amount of γ-glutamylcysteine synthetase (γ-GCS, a key enzyme of GSH biosynthesis in liver from SF rats. In liver from SF rats, glycine also decreases the insulin-induced phosphorylation of insulin receptor substrate-1 (ISR-1 in serine residue and increases the phosphorylation of insulin receptor β-subunit (IR-β in tyrosine residue. Thus, supplementing diets with glycine to correct GSH deficiency and to reduce oxidative stress provides significant metabolic benefits to SF rats by improving insulin sensitivity.

The role of transient receptor potential channels in metabolic syndrome

DEFF Research Database (Denmark)

Liu, Daoyan; Zhu, Zhiming; Tepel, Martin

2008-01-01

Metabolic syndrome is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential (TRP...

Synthesis and distribution of N-benzyloxycarbonyl-[14C]-glycine, a lipophilic derivative of glycine

International Nuclear Information System (INIS)

Lambert, D.M.; Gallez, Bernard; Poupaert, J.H.

1995-01-01

N-benzyloxycarbonyl[ 14 C]-glycine, a lipophilic derivative of glycine exhibiting anticonvulsant properties, was prepared in one step from [U- 14 C] glycine and benzyl chloroformate in alkali medium. a comparative study of biodistribution was carried on mice between this compound and the parent amino-acid after intravenous administration. Dimethylsulfoxide was used as injection vehicle for N-benzyloxycarbonylglycine. The influence of this injection vehicle was studied comparing glycine injected in a saline solution and glycine co-administered with dimethylsulfoxide. No significant difference was found between these two treatments. Compared to glycine, N-benzyloxycarbonylglycine reached quickly the central nervous system and exhibited an enhanced brain penetration index, 13-fold superior to the parent aminoacid value. (Author)

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

Science.gov (United States)

Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

2017-01-01

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

Extrasynaptic glycine receptors of rodent dorsal raphe serotonergic neurons:a sensitive target for ethanol

OpenAIRE

Maguire, Edward P.; Mitchell, Elizabeth A.; Greig, Scott J.; Corteen, Nicole; Balfour, David J. K.; Swinny, Jerome; Lambert, Jeremy J.; Belelli, Delia

2014-01-01

Alcohol abuse is a significant medical and social problem. Several neurotransmitter systems are implicated in ethanol's actions, with certain receptors and ion channels emerging as putative targets. The dorsal raphe (DR) nucleus is associated with the behavioral actions of alcohol, but ethanol actions on these neurons are not well understood. Here, using immunohistochemistry and electrophysiology we characterize DR inhibitory transmission and its sensitivity to ethanol. DR neurons exhibit inh...

Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

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John G Yamauchi

2011-01-01

Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

FMRFamide-gated sodium channel and ASIC channels: a new class of ionotropic receptors for FMRFamide and related peptides.

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Lingueglia, Eric; Deval, Emmanuel; Lazdunski, Michel

2006-05-01

FMRFamide and related peptides typically exert their action through G-protein coupled receptors. However, two ionotropic receptors for these peptides have recently been identified. They are both members of the epithelial amiloride-sensitive Na+ channel and degenerin (ENaC/DEG) family of ion channels. The invertebrate FMRFamide-gated Na+ channel (FaNaC) is a neuronal Na+-selective channel which is directly gated by micromolar concentrations of FMRFamide and related tetrapeptides. Its response is fast and partially desensitizing, and FaNaC has been proposed to participate in peptidergic neurotransmission. On the other hand, mammalian acid-sensing ion channels (ASICs) are not gated but are directly modulated by FMRFamide and related mammalian peptides like NPFF and NPSF. ASICs are activated by external protons and are therefore extracellular pH sensors. They are expressed both in the central and peripheral nervous system and appear to be involved in many physiological and pathophysiological processes such as hippocampal long-term potentiation and defects in learning and memory, acquired fear-related behavior, retinal function, brain ischemia, pain sensation in ischemia and inflammation, taste perception, hearing functions, and mechanoperception. The potentiation of ASIC activity by endogenous RFamide neuropeptides probably participates in the response to noxious acidosis in sensory and central neurons. Available data also raises the possibility of the existence of still unknown FMRFamide related endogenous peptides acting as direct agonists for ASICs.

5-HT1A receptors modulate small-conductance Ca2+-activated K+ channels

DEFF Research Database (Denmark)

Grunnet, Morten; Jespersen, Thomas; Perrier, Jean-François

2004-01-01

Small-conductance calcium-activated potassium channels (SK) are responsible for the medium afterhyperpolarisation (mAHP) following action potentials in neurons. Here we tested the ability of serotonin (5-HT) to modulate the activity of SK channels by coexpressing 5-HT1A receptors with different...

The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures.

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Mayer, Mark L

2017-11-21

Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed. Published by Elsevier Inc.

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

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Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

SH Oxidation Stimulates Calcium Release Channels (Ryanodine Receptors From Excitable Cells

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CECILIA HIDALGO

2000-01-01

Full Text Available The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub µM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed

Synthesis and distribution of N-benzyloxycarbonyl-[{sup 14}C]-glycine, a lipophilic derivative of glycine

Energy Technology Data Exchange (ETDEWEB)

Lambert, D.M.; Gallez, Bernard; Poupaert, J.H. [Universite Catholique de Louvain, Brussels (Belgium). Dept. des Sciences Pharmaceutiques

1995-12-31

N-benzyloxycarbonyl[{sup 14}C]-glycine, a lipophilic derivative of glycine exhibiting anticonvulsant properties, was prepared in one step from [U-{sup 14}C] glycine and benzyl chloroformate in alkali medium. a comparative study of biodistribution was carried on mice between this compound and the parent amino-acid after intravenous administration. Dimethylsulfoxide was used as injection vehicle for N-benzyloxycarbonylglycine. The influence of this injection vehicle was studied comparing glycine injected in a saline solution and glycine co-administered with dimethylsulfoxide. No significant difference was found between these two treatments. Compared to glycine, N-benzyloxycarbonylglycine reached quickly the central nervous system and exhibited an enhanced brain penetration index, 13-fold superior to the parent aminoacid value. (Author).

Potentiation of gamma aminobutyric acid receptors (GABAAR by Ethanol: How are inhibitory receptors affected?

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Benjamin eFörstera

2016-05-01

Full Text Available In recent years there has been an increase in the understanding of ethanol actions on the type A -aminobutyric acid chloride channel (GABAAR, a member of the pentameric ligand gated ion channels (pLGICs. However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR, another member of the pLGIC family, as a suitable target for the development of new pharmacological tools.

Temporal alteration of spreading depression by the glycine transporter type-1 inhibitors NFPS and Org-24461 in chicken retina.

Science.gov (United States)

Kertesz, Szabolcs; Szabo, Geza; Udvari, Szabolcs; Levay, Gyorgy; Matyus, Peter; Harsing, Laszlo G

2013-01-25

We used isolated chicken retina to induce spreading depression by the glutamate receptor agonist N-methyl-d-aspartate. The N-methyl-d-aspartate-induced latency time of spreading depression was extended by the glycine(B) binding site competitive antagonist 7-chlorokynurenic acid. Addition of the glycine transporter type-1 inhibitors NFPS and Org-24461 reversed the inhibitory effect of 7-chlorokynurenic acid on N-methyl-d-aspartate-evoked spreading depression. The glycine uptake inhibitory activity of Org-24461, NFPS, and some newly synthesized analogs of NFPS was determined in CHO cells stably expressing human glycine transporter type-1b isoform. Compounds, which failed to inhibit glycine transporter type-1, also did not have effect on retinal spreading depression. These experiments indicate that the spreading depression model in chicken retina is a useful in vitro test to determine activity of glycine transporter type-1 inhibitors. In addition, our data serve further evidence for the role of glycine transporter type-1 in retinal neurotransmission and light processing. Copyright © 2012 Elsevier B.V. All rights reserved.

Do TRPC channels support working memory? Comparing modulations of TRPC channels and working memory through G-protein coupled receptors and neuromodulators.

Science.gov (United States)

Reboreda, Antonio; Theissen, Frederik M; Valero-Aracama, Maria J; Arboit, Alberto; Corbu, Mihaela A; Yoshida, Motoharu

2018-03-01

Working memory is a crucial ability we use in daily life. However, the cellular mechanisms supporting working memory still remain largely unclear. A key component of working memory is persistent neural firing which is believed to serve short-term (hundreds of milliseconds up to tens of seconds) maintenance of necessary information. In this review, we will focus on the role of transient receptor potential canonical (TRPC) channels as a mechanism underlying persistent firing. Many years of in vitro work have been suggesting a crucial role of TRPC channels in working memory and temporal association tasks. If TRPC channels are indeed a central mechanism for working memory, manipulations which impair or facilitate working memory should have a similar effect on TRPC channel modulation. However, modulations of working memory and TRPC channels were never systematically compared, and it remains unanswered whether TRPC channels indeed contribute to working memory in vivo or not. In this article, we review the effects of G-protein coupled receptors (GPCR) and neuromodulators, including acetylcholine, noradrenalin, serotonin and dopamine, on working memory and TRPC channels. Based on comparisons, we argue that GPCR and downstream signaling pathways that activate TRPC, generally support working memory, while those that suppress TRPC channels impair it. However, depending on the channel types, areas, and systems tested, this is not the case in all studies. Further work to clarify involvement of specific TRPC channels in working memory tasks and how they are affected by neuromodulators is still necessary in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

Science.gov (United States)

Yadav, Rajeev; Lu, H Peter

2018-03-28

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel

Science.gov (United States)

Li-Smerin, Yingying; Swartz, Kenton J.

2000-01-01

Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis. PMID:10828242

Direct Effect of Remifentanil and Glycine Contained in Ultiva® on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses.

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Makoto Sumie

Full Text Available Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord.We made patch-clamp recordings from substantia gelatinosa (SG neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli.Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine.We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous

Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

Science.gov (United States)

Bhattarai, Janardhan Prasad; Park, Soo Joung; Han, Seong Kyu

2013-01-01

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM) and almost completely blocked by strychnine (2 μM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABAA receptor (GABAAR)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons. PMID:24379976

Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

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Thi Thanh Hoang Nguyen

2013-01-01

Full Text Available The substantia gelatinosa (SG of the trigeminal subnucleus caudalis (Vc has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM and almost completely blocked by strychnine (2 μM, suggesting that taurine-mediated responses are via glycine receptor (GlyR activation. In addition, taurine (1 mM activated extrasynaptic GABAA receptor (GABAAR-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons.

Transient receptor potential (TRP) channels as drug targets for diseases of the digestive system

Science.gov (United States)

Holzer, Peter

2011-01-01

Approximately 20 of the 30 mammalian transient receptor potential (TRP) channel subunits are expressed by specific neurons and cells within the alimentary canal. They subserve important roles in taste, chemesthesis, mechanosensation, pain and hyperalgesia and contribute to the regulation of gastrointestinal motility, absorptive and secretory processes, blood flow, and mucosal homeostasis. In a cellular perspective, TRP channels operate either as primary detectors of chemical and physical stimuli, as secondary transducers of ionotropic or metabotropic receptors, or as ion transport channels. The polymodal sensory function of TRPA1, TRPM5, TRPM8, TRPP2, TRPV1, TRPV3 and TRPV4 enables the digestive system to survey its physical and chemical environment, which is relevant to all processes of digestion. TRPV5 and TRPV6 as well as TRPM6 and TRPM7 contribute to the absorption of Ca2+ and Mg2+, respectively. TRPM7 participates in intestinal pacemaker activity, and TRPC4 transduces muscarinic acetylcholine receptor activation to smooth muscle contraction. Changes in TRP channel expression or function are associated with a variety of diseases/disorders of the digestive system, notably gastro-esophageal reflux disease, inflammatory bowel disease, pain and hyperalgesia in heartburn, functional dyspepsia and irritable bowel syndrome, cholera, hypomagnesemia with secondary hypocalcemia, infantile hypertrophic pyloric stenosis, esophageal, gastrointestinal and pancreatic cancer, and polycystic liver disease. These implications identify TRP channels as promising drug targets for the management of a number of gastrointestinal pathologies. As a result, major efforts are put into the development of selective TRP channel agonists and antagonists and the assessment of their therapeutic potential. PMID:21420431

Glycine regulates the production of pro-inflammatory cytokines in lean and monosodium glutamate-obese mice.

Science.gov (United States)

Alarcon-Aguilar, F J; Almanza-Perez, Julio; Blancas, Gerardo; Angeles, Selene; Garcia-Macedo, Rebeca; Roman, Ruben; Cruz, Miguel

2008-12-03

Fat tissue plays an important role in the regulation of inflammatory processes. Increased visceral fat has been associated with a higher production of cytokines that triggers a low-grade inflammatory response, which eventually may contribute to the development of insulin resistance. In the present study, we investigated whether glycine, an amino acid that represses the expression in vitro of pro-inflammatory cytokines in Kupffer and 3T3-L1 cells, can affect in vivo cytokine production in lean and monosodium glutamate-induced obese mice (MSG/Ob mice). Our data demonstrate that glycine treatment in lean mice suppressed TNF-alpha transcriptional expression in fat tissue, and serum protein levels of IL-6 were suppressed, while adiponectin levels were increased. In MSG/Ob mice, glycine suppressed TNF-alpha and IL-6 gene expression in fat tissue and significantly reduced protein levels of IL-6, resistin and leptin. To determine the role of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the modulation of this inflammatory response evoked by glycine, we examined its expression levels in fat tissue. Glycine clearly increased PPAR-gamma expression in lean mice but not in MSG/Ob mice. Finally, to identify alterations in glucose metabolism by glycine, we also examined insulin levels and other biochemical parameters during an oral glucose tolerance test. Glycine significantly reduced glucose tolerance and raised insulin levels in lean but not in obese mice. In conclusion, our findings suggest that glycine suppresses the pro-inflammatory cytokines production and increases adiponectin secretion in vivo through the activation of PPAR-gamma. Glycine might prevent insulin resistance and associated inflammatory diseases.

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels.

Science.gov (United States)

Shang, Lijun; Tucker, Stephen J

2008-02-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.

Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

DEFF Research Database (Denmark)

Rivera-Acevedo, Ricardo E; Pless, Stephan Alexander; Schwarz, Stephan K W

2013-01-01

Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing...

P2Y2 and P2Y4 receptors regulate pancreatic Ca²+-activated K+ channels differently

DEFF Research Database (Denmark)

Klærke, Susanne Edeling Hede; Amstrup, Jan; Klærke, Dan Arne

2005-01-01

Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, we have shown that ATP modulates epithelial K+ channels via purinergic receptors, most likely the P2Y2 and P2Y4 receptors, but the identity of the involved K+ channels was not cle...

Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs.

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Shigeru Saito

2011-04-01

Full Text Available Transient Receptor Potential (TRP channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.

Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

DEFF Research Database (Denmark)

Thilo, Florian; Liu, Ying; Krueger, Katharina

2012-01-01

The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...... to control conditions. We therefore hypothesize that cysteine residues increase TRPC6 channel protein expression in humans....

Receptor model for the molecular basis of tissue selectivity of 1,4-dihydropyridine calcium channel drugs

Science.gov (United States)

Langs, David A.; Strong, Phyllis D.; Triggle, David J.

1990-09-01

Our analysis of the solid state conformations of nifedipine [dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinecarboxylate] and its 1,4-dihydropyridine (1,4-DHP) analogues produced a cartoon description of the important interactions between these drugs and their voltage-dependent calcium channel receptor. In the present study a molecular-level detailed model of the 1,4-DHP receptor binding site has been built from the published amino acid sequence of the 215-1 subunit of the voltage-dependent calcium channel isolated from rabbit skeletal muscle transverse tubule membranes. The voltage-sensing component of the channel described in this work differs from others reported for the homologous sodium channel in that it incorporates a water structure and a staggered, rather than eclipsed, hydrogen bonded S4 helix conformation. The major recognition surfaces of the receptor lie in helical grooves on the S4 or voltagesensing α-helix that is positioned in the center of the bundle of transmembrane helices that define each of the four calcium channel domains. Multiple binding clefts defined by Arg-X-X-Arg-P-X-X-S `reading frames' exist on the S4 strand. The tissue selectivity of nifedipine and its analogues may arise, in part, from conservative changes in the amino acid residues at the P and S positions of the reading frame that define the ester-binding regions of receptors from different tissues. The crystal structures of two tissue-selective nifedipine analogues, nimodipine [isopropyl (2-methoxyethyl) 1,4-dihydro-2,6- dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] and nitrendipine [ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] are reported. Nimodipine was observed to have an unusual ester side chain conformation that enhances the fit to the proposed ester-sensing region of the receptor.

Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM

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Nigel Unwin

2017-07-01

Full Text Available Rapid communication at the chemical synapse depends on the action of ion channels residing in the postsynaptic membrane. The channels open transiently upon the binding of a neurotransmitter released from the presynaptic nerve terminal, eliciting an electrical response. Membrane lipids also play a vital but poorly understood role in this process of synaptic transmission. The present study examines the lipid distribution around nicotinic acetylcholine (ACh receptors in tubular vesicles made from postsynaptic membranes of the Torpedo ray, taking advantage of the recent advances in cryo-EM. A segregated distribution of lipid molecules is found in the outer leaflet of the bilayer. Apparent cholesterol-rich patches are located in specific annular regions next to the transmembrane helices and also in a more extended `microdomain' between the apposed δ subunits of neighbouring receptors. The particular lipid distribution can be interpreted straightforwardly in relation to the gating movements revealed by an earlier time-resolved cryo-EM study, in which the membranes were exposed briefly to ACh. The results suggest that in addition to stabilizing the protein, cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel.

Increased migration of monocytes in essential hypertension is associated with increased transient receptor potential channel canonical type 3 channels

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Zhao, Zhigang; Ni, Yinxing; Chen, Jing

2012-01-01

Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performe...

Dynamics of receptor-operated Ca2+ Currents Through TRPC Channels Controlled via the PI(4,5P2-PLC Signaling Pathway

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Masayuki X Mori

2015-02-01

Full Text Available Transient receptor potential canonical (TRPC channels are Ca2+-permeable, nonselective cation channels that carry receptor-operated Ca2+ currents (ROCs triggered by receptor-induced, phospholipase C (PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5P2. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5P2 and the possible role of PI(4,5P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5P2 using a model simulation that could explain the complicated effects of PI(4,5P2 on different ROCs.

Dynamics of receptor-operated Ca(2+) currents through TRPC channels controlled via the PI(4,5)P2-PLC signaling pathway.

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Mori, Masayuki X; Itsuki, Kyohei; Hase, Hideharu; Sawamura, Seishiro; Kurokawa, Tatsuki; Mori, Yasuo; Inoue, Ryuji

2015-01-01

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.

Formation of intercalation compound of kaolinite-glycine via displacing guest water by glycine.

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Zheng, Wan; Zhou, Jing; Zhang, Zhenqian; Chen, Likun; Zhang, Zhongfei; Li, Yong; Ma, Ning; Du, Piyi

2014-10-15

The kaolinite-glycine intercalation compound was successfully formed by displacing intercalated guest water molecules in kaolinite hydrate as a precursor. The microstructure of the compound was characterized by X-ray diffraction, Fourier Transform Infrared Spectroscopy and Scanning Electron Microscope. Results show that glycine can only be intercalated into hydrated kaolinite to form glycine-kaolinite by utilizing water molecules as a transition phase. The intercalated glycine molecules were squeezed partially into the ditrigonal holes in the silicate layer, resulting in the interlayer distance of kaolinite reaching 1.03nm. The proper intercalation temperature range was between 20°C and 80°C. An intercalation time of 24h or above was necessary to ensure the complete formation of kaolinite-glycine. The highest intercalation degree of about 84% appeared when the system was reacted at the temperature of 80°C for 48h. There were two activation energies for the intercalation of glycine into kaolinite, one being 21kJ/mol within the temperature range of 20-65°C and the other 5.8kJ/mol between 65°C and 80°C. The intercalation degree (N) and intercalation velocity (v) of as a function of intercalation time (t) can be empirically expressed as N=-79.35e(-)(t)(/14.8)+80.1 and v=5.37e(-)(t)(/14.8), respectively. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of ionotropic GABA, glutamate and glycine receptors in the tonic and reflex control of cardiac vagal outflow in the rat

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Goodchild Ann K

2010-10-01

Full Text Available Abstract Background Cardiac vagal preganglionic neurons (CVPN are responsible for the tonic, reflex and respiratory modulation of heart rate (HR. Although CVPN receive GABAergic and glutamatergic inputs, likely involved in respiratory and reflex modulation of HR respectively, little else is known regarding the functions controlled by ionotropic inputs. Activation of g-protein coupled receptors (GPCR alters these inputs, but the functional consequence is largely unknown. The present study aimed to delineate how ionotropic GABAergic, glycinergic and glutamatergic inputs contribute to the tonic and reflex control of HR and in particular determine which receptor subtypes were involved. Furthermore, we wished to establish how activation of the 5-HT1A GPCR affects tonic and reflex control of HR and what ionotropic interactions this might involve. Results Microinjection of the GABAA antagonist picrotoxin into CVPN decreased HR but did not affect baroreflex bradycardia. The glycine antagonist strychnine did not alter HR or baroreflex bradycardia. Combined microinjection of the NMDA antagonist, MK801, and AMPA antagonist, CNQX, into CVPN evoked a small bradycardia and abolished baroreflex bradycardia. MK801 attenuated whereas CNQX abolished baroreceptor bradycardia. Control intravenous injections of the 5-HT1A agonist 8-OH-DPAT evoked a small bradycardia and potentiated baroreflex bradycardia. These effects were still observed following microinjection of picrotoxin but not strychnine into CVPN. Conclusions We conclude that activation of GABAA receptors set the level of HR whereas AMPA to a greater extent than NMDA receptors elicit baroreflex changes in HR. Furthermore, activation of 5-HT1A receptors evokes bradycardia and enhances baroreflex changes in HR due to interactions with glycinergic neurons involving strychnine receptors. This study provides reference for future studies investigating how diseases alter neurochemical inputs to CVPN.

Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis

International Nuclear Information System (INIS)

Castric, P.A.

1977-01-01

Hydrogen cyanide (HCN) production by Pseudomonas aeruginosa in a synthetic medium is stimulated by the presence of glycine. Methionine enhances this stimulation but will not substitute for glycine as a stimulator of cyanogenesis. Threonine and phenylalanine are effective substitutes for glycine in the stimulation of HCN production. Glycine, threonine, and serine are good radioisotope precursors of HCN, but methionine and phenylalanine are not. Cell extracts of P. aeruginosa convert [ 14 C]threonine to [ 14 C]glycine. H14CN is produced with low dilution of label from either [1- 14 C]glycine or [2- 14 C]glycine, indicating a randomization of label either in the primary or secondary metabolism of glycine. When whole cells were fed [1,2- 14 C]glycine, cyanide and bicarbonate were the only radioactive extracellular products observed

Glycine Transporter Inhibitor Attenuates the Psychotomimetic Effects of Ketamine in Healthy Males: Preliminary Evidence

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D'Souza, Deepak Cyril; Singh, Nagendra; Elander, Jacqueline; Carbuto, Michelle; Pittman, Brian; de Haes, Joanna Udo; Sjogren, Magnus; Peeters, Pierre; Ranganathan, Mohini; Schipper, Jacques

2012-01-01

Enhancing glutamate function by stimulating the glycine site of the NMDA receptor with glycine, -serine, or with drugs that inhibit glycine reuptake may have therapeutic potential in schizophrenia. The effects of a single oral dose of cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-ylmethyl) amino-methylcarboxylic acid hydrochloride (Org 25935), a glycine transporter-1 (GlyT1) inhibitor, and placebo pretreatment on ketamine-induced schizophrenia-like psychotic symptoms, perceptual alterations, and subjective effects were evaluated in 12 healthy male subjects in a randomized, counter-balanced, within-subjects, crossover design. At 2.5 h after administration of the Org 25935 or placebo, subjects received a ketamine bolus and constant infusion lasting 100 min. Psychotic symptoms, perceptual, and a number of subjective effects were assessed repeatedly before, several times during, and after completion of ketamine administration. A cognitive battery was administered once per test day. Ketamine produced behavioral, subjective, and cognitive effects consistent with its known effects. Org 25935 reduced the ketamine-induced increases in measures of psychosis (Positive and Negative Syndrome Scale (PANSS)) and perceptual alterations (Clinician Administered Dissociative Symptoms Scale (CADSS)). The magnitude of the effect of Org 25935 on ketamine-induced increases in Total PANSS and CADSS Clinician-rated scores was 0.71 and 0.98 (SD units), respectively. None of the behavioral effects of ketamine were increased by Org 25935 pretreatment. Org 25935 worsened some aspects of learning and delayed recall, and trended to improve choice reaction time. This study demonstrates for the first time in humans that a GlyT1 inhibitor reduces the effects induced by NMDA receptor antagonism. These findings provide preliminary support for further study of the antipsychotic potential of GlyT1 inhibitors. PMID:22113087

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium

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Adela Banciu

2018-05-01

Full Text Available Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1, estrogen receptors (ESR1, ESR2, GPR30, and nuclear receptor coactivator 3 (NCOA3. Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM antagonized the effect of BAY K8644 (2.5 or 5 µM in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

Science.gov (United States)

Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

2018-05-09

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine

Polymodal Transient Receptor Potential Vanilloid (TRPV Ion Channels in Chondrogenic Cells

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Csilla Szűcs Somogyi

2015-08-01

Full Text Available Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

76 FR 8771 - Glycine From China

Science.gov (United States)

2011-02-15

... INTERNATIONAL TRADE COMMISSION [Investigation No. 731-TA-718 (Third Review)] Glycine From China... order on glycine from China. SUMMARY: The Commission hereby gives notice that it will proceed with a... determine whether revocation of the antidumping duty order on glycine from China would be likely to lead to...

76 FR 55109 - Glycine From China

Science.gov (United States)

2011-09-06

... INTERNATIONAL TRADE COMMISSION [Investigation No. 731-TA-718 (Third Review)] Glycine From China... U.S.C. 1675(c)), that revocation of the antidumping duty order on glycine from China would be likely... contained in USITC Publication 4255 (August 2011), entitled Glycine from China: Investigation No. 731-TA-718...

Utilization of [1-14C]carbon of glycine of high glycine diet fed young and old rats

International Nuclear Information System (INIS)

Petzke, K.J.; Albrecht, V.; Medovar, B.Ya.; Pisarczuk, K.L.; Grigorov, Yu.G.

1987-01-01

The incorporation of radioactivity from [1- 14 C]glycine was studied in various organ (serum, liver, muscle) fractions (acid soluble, proteins, lipids, liver glycogen) and carbon dioxide in rats fed with isonitrogenous isocaloric purfied diets. The diets contained 30% casein (control), gelatin (exchange of half of the 30% casein) or glycine (corresponding level of glycine in relation to the gelatin diet). The incorporation of radioactivity into proteins was reduced by feeding high glycine diets in young (20-weeks-old) and old (18-month-old) rats in relation to the control diet. The modifications of the results for old animals may be partially explained on the base of a reduced protein turnover rate and adaptation to a high gelatin (glycine) diet. (author)

Glycine serine interconversion in the rooster

International Nuclear Information System (INIS)

Sugahara, Michihiro; Kandatsu, Makoto

1976-01-01

Serine was isolated by the column chromatography from the hydrolyzates of proteins of the serum, the liver and the pectoral muscle which were obtained from the roosters fed a diet containing 2- 14 C glycine for 16 - 17 days. The carbon chain of serine was cut off by treating with sodium periodate. The specific activity of each carbon (as barium carbonate) was estimated. Carboxyl carbon had little radioactivity. The specific activity of hydroxymethyl carbon was 10 - 19% of that of methylene carbon. Glycine isolated from the same hydrolyzates was degraded by ninhydrin oxidation. Formaldehyde produced from 2-C was oxidized to carbon dioxide by treating with mercuric chloride. Carboxyl carbon had little radioactivity. The specific activities of 2-C of glycine and 2-C of serine in the same tissue protein were compared. The ratio of serine 2-C/glycine 2-C was between 0.7 - 1.5. These results seem to indicate that glycine directly converts to serine in the rooster. The quantitative significance of the pathways of glycine (serine) biosynthesis is discussed. (auth.)

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...

A polycystin-type transient receptor potential (Trp channel that is activated by ATP

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David Traynor

2017-02-01

Full Text Available ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.

Review article: transient receptor potential channels as possible therapeutic targets in irritable bowel syndrome.

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Beckers, A B; Weerts, Z Z R M; Helyes, Z; Masclee, A A M; Keszthelyi, D

2017-11-01

Abdominal pain in irritable bowel syndrome (IBS) remains challenging to treat effectively. Researchers have attempted to elucidate visceral nociceptive processes in order to guide treatment development. Transient receptor potential (TRP) channels have been implied in the generation (TRPV1, TRPV4, TRPA1) and inhibition (TRPM8) of visceral pain signals. Pathological changes in their functioning have been demonstrated in inflammatory conditions, and appear to be present in IBS as well. To provide a comprehensive review of the current literature on TRP channels involved in visceral nociception. In particular, we emphasise the clinical implications of these nociceptors in the treatment of IBS. Evidence to support this review was obtained from an electronic database search via PubMed using the search terms "visceral nociception," "visceral hypersensitivity," "irritable bowel syndrome" and "transient receptor potential channels." After screening the abstracts the articles deemed relevant were cross-referenced for additional manuscripts. Recent studies have resulted in significant advances in our understanding of TRP channel mediated visceral nociception. The diversity of TRP channel sensitization pathways is increasingly recognised. Endogenous TRP agonists, including poly-unsaturated fatty acid metabolites and hydrogen sulphide, have been implied in augmented visceral pain generation in IBS. New potential targets for treatment development have been identified (TRPA1 and TRPV4,) and alternative means of affecting TRP channel signalling (partial antagonists, downstream targeting and RNA-based therapy) are currently being explored. The improved understanding of mechanisms involved in visceral nociception provides a solid basis for the development of new treatment strategies for abdominal pain in IBS. © 2017 John Wiley & Sons Ltd.

Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

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Paul C.P. Curtin

2015-03-01

Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

A ligand channel through the G protein coupled receptor opsin.

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Peter W Hildebrand

Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

Increased transient receptor potential canonical type 3 channels in vasculature from hypertensive rats

DEFF Research Database (Denmark)

Liu, Daoyan; Yang, Dachun; He, Hongbo

2009-01-01

We tested the hypothesis that transient receptor potential canonical type 3 (TRPC3) channels are increased in vascular smooth muscle cells and aortic tissue from spontaneously hypertensive rats (SHR) compared with normotensive Wistar Kyoto rats. Expression of TRPC3 was analyzed by immunohistochem...

Difference in brain distributions of carbon 11-labeled 4-hydroxy-2(1H)-quinolones as PET radioligands for the glycine-binding site of the NMDA ion channel

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Fuchigami, Takeshi [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Haradahira, Terushi [Molecular Imaging Center, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555 (Japan)], E-mail: terushi@niu.ac.jp; Fujimoto, Noriko [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Okauchi, Takashi; Maeda, Jun; Suzuki, Kazutoshi; Suhara, Tetsuya [Molecular Imaging Center, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555 (Japan); Yamamoto, Fumihiko; Sasaki, Shigeki; Mukai, Takahiro [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan); Yamaguchi, Hiroshi [Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Ogawa, Mikako [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Magata, Yasuhiro [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Maeda, Minoru [Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Japan)

2008-02-15

High-affinity iodine- and ethyl-C-5 substituted analogs of 4-hydroxy-3-(3-[{sup 11}C]methoxyphenyl)-2(1H)-quinolone ([{sup 11}C]4HQ) were synthesized as new positron emission tomography radioligands for the glycine-binding sites of the N-methyl-D-aspartate (NMDA) ion channel. Although both radioligands showed high in vitro specific binding to rat brain slices, their binding characteristics were quite different from each other. 5-Ethyl-[{sup 11}C]4HQ (5Et-[{sup 11}C]4HQ) showed higher in vitro binding in the forebrain regions than in the cerebellum, bindings that were strongly inhibited by both glycine-site agonists and antagonists. In contrast, 5-iodo-[{sup 11}C]4HQ (5I-[{sup 11}C]4HQ) showed a homogeneous in vitro binding throughout the brain, which was inhibited by antagonists but not by agonists. This difference in in vitro binding between 5Et-[{sup 11}C]4HQ and 5I-[{sup 11}C]4HQ was quite similar to that previously observed between [{sup 11}C]L-703,717 and [{sup 11}C]4HQ, both glycine-site antagonists. In vivo brain uptakes of these {sup 11}C-labeled 4-hydroxyquinolones were examined in mice. Initial brain uptakes of 5Et- and 5I-[{sup 11}C]4HQ at 1 min after intravenous injections were comparable to that of [{sup 11}C]4HQ, but they were 1.3-2.1 times higher than that of [{sup 11}C]L-703,717. The treatment with an anticoagulant, warfarin, only slightly increased the initial uptakes of [{sup 11}C]4HQ and 5Et-[{sup 11}C]4HQ in contrast to [{sup 11}C]L-703,717. The in vivo regional brain distributions were slightly different between the two radioligands. Pretreatment with nonradioactive ligand (2 mg/kg) slightly inhibited the binding of 5Et-[{sup 11}C]4HQ (16-36% inhibition) but not that of 5I-[{sup 11}C]4HQ. In this study, it was found that a small structural change in [{sup 11}C]4HQ resulted in a major change in binding characteristics and distributions, suggesting the existence of two binding sites for [{sup 11}C]4-hydroxyquinolones on the NMDA ion channel

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

Energy Technology Data Exchange (ETDEWEB)

Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

2008-10-27

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

Science.gov (United States)

Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

2002-04-01

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

Molecular mechanisms of Cys-loop ion channel receptor modulation by ivermectin

DEFF Research Database (Denmark)

Lynagh, Timothy; Lynch, Joseph W.

2012-01-01

Ivermectin is an anthelmintic drug that works by inhibiting neuronal activity and muscular contractility in arthropods and nematodes. It works by activating glutamate-gated chloride channels (GluClRs) at nanomolar concentrations. These receptors, found exclusively in invertebrates, belong to the ...... to the neurotransmitter binding site, thus suggesting a mechanism by which ivermectin potentiates neurotransmitter-gated currents. Together, this information provides new insights into the mechanisms of action of this important drug.......) to its site. Several lines of evidence suggest that ivermectin opens the channel pore via a structural change distinct from that induced by the neurotransmitter agonist. Conformational changes occurring at locations distant from the pore can be probed using voltage-clamp fluorometry (VCF), a technique...

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1.

Science.gov (United States)

Chang, Yiming; Schlenstedt, Gabriel; Flockerzi, Veit; Beck, Andreas

2010-05-17

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A decline in transcript abundance for Heterodera glycines homologs of Caenorhabditis elegans uncoordinated genes accompanies its sedentary parasitic phase

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Overall Christopher C

2007-04-01

Full Text Available Abstract Background Heterodera glycines (soybean cyst nematode [SCN], the major pathogen of Glycine max (soybean, undergoes muscle degradation (sarcopenia as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87 (calponin during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia. Results We developed a bioinformatics database that compares expressed sequence tag (est and genomic data of C. elegans and H. glycines (CeHg database. We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin, unc-89, unc-15 (paromyosin, unc-27 (troponin I, unc-54 (myosin, and the potassium channel unc-110 (twk-18. Less reduction is observed for the focal adhesion complex gene Hg-unc-97. Conclusion The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.

Dual Gating Mechanism and Function of P2X7 Receptor Channels

Czech Academy of Sciences Publication Activity Database

Khadra, A.; Tomic, M.; Yan, Z.; Zemková, Hana; Sherman, A.; Stojilkovic, S. S.

2013-01-01

Roč. 104, č. 12 (2013), s. 2612-2621 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : purinergic P2X7 receptors * ATP-gated channels * BzATP * dilation * Markov -state model Subject RIV: ED - Physiology Impact factor: 3.832, year: 2013

Do glycine-extended hormone precursors have clinical significance?

DEFF Research Database (Denmark)

Rehfeld, Jens Frederik

2014-01-01

Half of the known peptide hormones are C-terminally amidated. Subsequent biogenesis studies have shown that the immediate precursor is a glycine-extended peptide. The clinical interest in glycine-extended hormones began in 1994, when it was suggested that glycine-extended gastrin stimulated cancer...... and clinical effects of glycine-extended precursors for most other amidated hormones than gastrin and cholecystokinin (CCK). The idea of glycine-extended peptides as independent messengers was interesting. But clinical science has to move ahead from ideas that cannot be supported at key points after decades...

Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

Science.gov (United States)

Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J; Buddenkotte, Jörg; Steinhoff, Martin

2012-04-01

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

Pharmacology of morphine and morphine-3-glucuronide at opioid, excitatory amino acid, GABA and glycine binding sites

Energy Technology Data Exchange (ETDEWEB)

Bartlett, S.E.; Smith, M.T. (Department of Pharmacy, The University of Queensland (Australia)); Dood, P.R. (Clinical Research Centre, Royal Brisbane Hospital Foundation, Brisbane (Australia))

1994-07-01

Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of [sup 3]H-MK801 (non-competitive antagonist) and [sup 125]I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of [sup 3]H-muscimol (GABA receptor agonist), [sup 3]H-diazepam and [sup 3]H-flunitraxepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABA[sub A] receptors. Morphine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of [sup 3]H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors. (au) (30 refs.).

Allosteric regulation of the P2X4 receptor channel pore dilation

Czech Academy of Sciences Publication Activity Database

Zemková, Hana; Khadra, A.; Rokic, Milos Boro; Tvrdoňová, Vendula; Sherman, A.; Stojilkovic, S. S.

2015-01-01

Roč. 467, č. 4 (2015), s. 713-726 ISSN 0031-6768 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ATP * purinergic receptor channel * ivermectin * pore dilation * Markov state model Subject RIV: ED - Physiology Impact factor: 3.654, year: 2015

Mechanics of channel gating of the nicotinic acetylcholine receptor.

Directory of Open Access Journals (Sweden)

Xinli Liu

2008-01-01

Full Text Available The nicotinic acetylcholine receptor (nAChR is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD and steered rotation molecular dynamics (SRMD simulations have been conducted on the cryo-electron microscopy (cryo-EM structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC, transmembrane (TM, and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA. To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O C motion. The present MD simulations explore the structural dynamics of the receptor under its

Canonical transient receptor potential channel 2 (TRPC2): old name-new games. Importance in regulating of rat thyroid cell physiology.

Science.gov (United States)

Törnquist, Kid; Sukumaran, Pramod; Kemppainen, Kati; Löf, Christoffer; Viitanen, Tero

2014-11-01

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

21 CFR 520.550 - Dextrose/glycine/electrolyte.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Dextrose/glycine/electrolyte. 520.550 Section 520...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.550 Dextrose/glycine..., potassium citrate 0.12 gram, aminoacetic acid (glycine) 6.36 grams, and dextrose 44.0 grams. (b) Sponsor...

Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage.

Science.gov (United States)

Cisneros-Mejorado, Abraham; Gottlieb, Miroslav; Cavaliere, Fabio; Magnus, Tim; Koch-Nolte, Friederich; Scemes, Eliana; Pérez-Samartín, Alberto; Matute, Carlos

2015-05-01

The role of P2X7 receptors and pannexin-1 channels in ischemic damage remains controversial. Here, we analyzed their contribution to postanoxic depolarization after ischemia in cultured neurons and in brain slices. We observed that pharmacological blockade of P2X7 receptors or pannexin-1 channels delayed the onset of postanoxic currents and reduced their slope, and that simultaneous inhibition did not further enhance the effects of blocking either one. These results were confirmed in acute cortical slices from P2X7 and pannexin-1 knockout mice. Oxygen-glucose deprivation in cortical organotypic cultures caused neuronal death that was reduced with P2X7 and pannexin-1 blockers as well as in organotypic cultures derived from mice lacking P2X7 and pannexin 1. Subsequently, we used transient middle cerebral artery occlusion to monitor the neuroprotective effect of those drugs in vivo. We found that P2X7 and pannexin-1 antagonists, and their ablation in knockout mice, substantially attenuated the motor symptoms and reduced the infarct volume to ~50% of that in vehicle-treated or wild-type animals. These results show that P2X7 receptors and pannexin-1 channels are major mediators of postanoxic depolarization in neurons and of brain damage after ischemia, and that they operate in the same deleterious signaling cascade leading to neuronal and tissue demise.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels

Science.gov (United States)

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-01-01

Store-operated Ca2+ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca2+ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca2+]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. PMID:27129253

Glycine

DEFF Research Database (Denmark)

Sabin, John R.; Oddershede, Jens; Sauer, Stephan P. A.

2013-01-01

With the advent of the use of precise ion accelerators for medical purposes, it becomes ever more important to understand the interaction of biomolecules with fast ions.  Glycine is both a protein component and a model biomolecule, and is thus an important test system.    In this report, we discu...

The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster.

Science.gov (United States)

Wolfgang, Werner; Simoni, Alekos; Gentile, Carla; Stanewsky, Ralf

2013-10-07

Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber ('time giver') and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16-20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine.

Science.gov (United States)

Yasukochi, Midori; Uehara, Akira; Kobayashi, Sei; Berlin, Joshua R

2003-03-01

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.

Glycine phases formed from frozen aqueous solutions: Revisited

Energy Technology Data Exchange (ETDEWEB)

Surovtsev, N. V. [Institute of Automation and Electrometry, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Novosibirsk State University, Novosibirsk 630090 (Russian Federation); Adichtchev, S. V.; Malinovsky, V. K. [Institute of Automation and Electrometry, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Ogienko, A. G.; Manakov, A. Yu. [Novosibirsk State University, Novosibirsk 630090 (Russian Federation); Nikolaev Institute of Inorganic Chemistry, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Drebushchak, V. A. [Novosibirsk State University, Novosibirsk 630090 (Russian Federation); Institute of Geology and Mineralogy, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Ancharov, A. I.; Boldyreva, E. V. [Novosibirsk State University, Novosibirsk 630090 (Russian Federation); Institute of Solid Chemistry and Mechanochemistry, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation); Yunoshev, A. S. [Novosibirsk State University, Novosibirsk 630090 (Russian Federation); Lavrentiev Institute of Hydrodynamics, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090 (Russian Federation)

2012-08-14

Glycine phases formed when aqueous solutions were frozen and subsequently heated under different conditions were studied by Raman scattering, x-ray diffraction, and differential scanning calorimetry (DSC) techniques. Crystallization of ice I{sub h} was observed in all the cases. On cooling at the rates of 0.5 K/min and 5 K/min, glassy glycine was formed as an intermediate phase which lived about 1 min or less only, and then transformed into {beta}-polymorph of glycine. Quench cooling of glycine solutions (15% w/w) in liquid nitrogen resulted in the formation of a mixture of crystalline water ice I{sub h} and a glassy glycine, which could be preserved at cryogenic temperatures (80 K) for an indefinitely long time. This mixture remained also quite stable for some time after heating above the cryogenic temperature. Subsequent heating under various conditions resulted in the transformation of the glycine glass into an unknown crystalline phase (glycine 'X-phase') at 209-216 K, which at 218-226 K transformed into {beta}-polymorph of glycine. The 'X-phase' was characterized by Raman spectroscopy; it could be obtained in noticeable amounts using a special preparation technique and tentatively characterized by x-ray powder diffraction (P2, a= 6.648 A, b= 25.867 A, c= 5.610 A, {beta}= 113.12 Masculine-Ordinal-Indicator ); the formation of 'X-phase' from the glycine glassy phase and its transformation into {beta}-polymorph were followed by DSC. Raman scattering technique with its power for unambiguous identification of the crystalline and glassy polymorphs without limitation on the crystallite size helped us to follow the phase transformations during quenching, heating, and annealing. The experimental findings are considered in relation to the problem of control of glycine polymorphism on crystallization.

Glycine phases formed from frozen aqueous solutions: Revisited

Science.gov (United States)

Surovtsev, N. V.; Adichtchev, S. V.; Malinovsky, V. K.; Ogienko, A. G.; Drebushchak, V. A.; Manakov, A. Yu.; Ancharov, A. I.; Yunoshev, A. S.; Boldyreva, E. V.

2012-08-01

Glycine phases formed when aqueous solutions were frozen and subsequently heated under different conditions were studied by Raman scattering, x-ray diffraction, and differential scanning calorimetry (DSC) techniques. Crystallization of ice Ih was observed in all the cases. On cooling at the rates of 0.5 K/min and 5 K/min, glassy glycine was formed as an intermediate phase which lived about 1 min or less only, and then transformed into β-polymorph of glycine. Quench cooling of glycine solutions (15% w/w) in liquid nitrogen resulted in the formation of a mixture of crystalline water ice Ih and a glassy glycine, which could be preserved at cryogenic temperatures (80 K) for an indefinitely long time. This mixture remained also quite stable for some time after heating above the cryogenic temperature. Subsequent heating under various conditions resulted in the transformation of the glycine glass into an unknown crystalline phase (glycine "X-phase") at 209-216 K, which at 218-226 K transformed into β-polymorph of glycine. The "X-phase" was characterized by Raman spectroscopy; it could be obtained in noticeable amounts using a special preparation technique and tentatively characterized by x-ray powder diffraction (P2, a = 6.648 Å, b = 25.867 Å, c = 5.610 Å, β = 113.12°); the formation of "X-phase" from the glycine glassy phase and its transformation into β-polymorph were followed by DSC. Raman scattering technique with its power for unambiguous identification of the crystalline and glassy polymorphs without limitation on the crystallite size helped us to follow the phase transformations during quenching, heating, and annealing. The experimental findings are considered in relation to the problem of control of glycine polymorphism on crystallization.

Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses.

Science.gov (United States)

Cash, D J; Subbarao, K

1987-12-01

The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

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Morgan, Peter J; Hübner, Rayk; Rolfs, Arndt; Frech, Moritz J

2013-09-15

Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

Multifarious Beneficial Effect of Nonessential Amino Acid, Glycine: A Review

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Meerza Abdul Razak

2017-01-01

Full Text Available Glycine is most important and simple, nonessential amino acid in humans, animals, and many mammals. Generally, glycine is synthesized from choline, serine, hydroxyproline, and threonine through interorgan metabolism in which kidneys and liver are the primarily involved. Generally in common feeding conditions, glycine is not sufficiently synthesized in humans, animals, and birds. Glycine acts as precursor for several key metabolites of low molecular weight such as creatine, glutathione, haem, purines, and porphyrins. Glycine is very effective in improving the health and supports the growth and well-being of humans and animals. There are overwhelming reports supporting the role of supplementary glycine in prevention of many diseases and disorders including cancer. Dietary supplementation of proper dose of glycine is effectual in treating metabolic disorders in patients with cardiovascular diseases, several inflammatory diseases, obesity, cancers, and diabetes. Glycine also has the property to enhance the quality of sleep and neurological functions. In this review we will focus on the metabolism of glycine in humans and animals and the recent findings and advances about the beneficial effects and protection of glycine in different disease states.

Coarse architecture of the transient receptor potential vanilloid 1 (TRPV1) ion channel determined by fluorescence resonance energy transfer.

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De-la-Rosa, Víctor; Rangel-Yescas, Gisela E; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara; Islas, León D

2013-10-11

The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane.

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase

OpenAIRE

Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

2016-01-01

Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and wheth...

Canonical Transient Receptor Potential Channels and Their Link with Cardio/Cerebro-Vascular Diseases.

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Xiao, Xiong; Liu, Hui-Xia; Shen, Kuo; Cao, Wei; Li, Xiao-Qiang

2017-09-01

The canonical transient receptor potential channels (TRPCs) constitute a series of nonselective cation channels with variable degrees of Ca 2+ selectivity. TRPCs consist of seven mammalian members, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7, which are further divided into four subtypes, TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7. These channels take charge of various essential cell functions such as contraction, relaxation, proliferation, and dysfunction. This review, organized into seven main sections, will provide an overview of current knowledge about the underlying pathogenesis of TRPCs in cardio/cerebrovascular diseases, including hypertension, pulmonary arterial hypertension, cardiac hypertrophy, atherosclerosis, arrhythmia, and cerebrovascular ischemia reperfusion injury. Collectively, TRPCs could become a group of drug targets with important physiological functions for the therapy of human cardio/cerebro-vascular diseases.

Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs

DEFF Research Database (Denmark)

Teilmann, Stefan C.; Byskov, Anne Grete; Pedersen, Per Amstrup

2005-01-01

We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures...... of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both...... polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high...

Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

NARCIS (Netherlands)

Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

Transcriptomic Analysis Of Purified Embryonic Neural Stem Cells From Zebrafish Embryos Reveals Signalling Pathways Involved In Glycine-dependent Neurogenesis

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Eric eSAMARUT

2016-03-01

Full Text Available How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signalling that promotes calcium transients in neural stem cells (NSCs and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting (FACS from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signalling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signalling pathways (signalling by calcium, TGF-beta, sonic hedgehog, Wnt and p53-related apoptosis that are likely to mediate the promotion of neurogenesis by glycine.

Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

Energy Technology Data Exchange (ETDEWEB)

Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

1990-05-01

Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

Different forms of glycine- and GABAA-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

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Brichta Alan M

2009-11-01

Full Text Available Abstract Background Neurons in superficial (SDH and deep (DDH laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR and GABAA-receptors (GABAARs contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood. Methods and Results Here we compare fast inhibitory synaptic transmission in mouse (P17-37 SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C. GlyR-mediated mIPSCs were detected in 74% (25/34 and 94% (25/27 of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18. Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each, decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms, and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18 and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8 and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11 in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA, had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM reduced GlyR-mediated mIPSC frequency in SDH

Sensitization of glycine (spectrophotometric read-out) dosimetric system using sorbitol

International Nuclear Information System (INIS)

Shinde, S.H.; Mukherjee, T.

2009-01-01

Glycine spectrophotometric read-out systems have a useful dose range of 15-4000 Gy. An attempt was made to sensitize it using sorbitol as a sensitizer. Optimum compositions of aqueous acidic solutions of ferrous ammonium sulphate-xylenol orange (XO), i.e. FX and sorbitol-ferrous ammonium sulphate-xylenol orange, i.e. SFX, for 400 mg of glycine, which gives maximum dosimetric response for any given dose, were established. Molar absorption coefficient values of ferric-XO-glycine complex, i.e. ε-values, were determined for glycine system in FX and SFX. These values were found to be 8410 and 15,000 m 2 mol -1 respectively, indicating that an enhancement or sensitivity factor of about 1.78 can be achieved by sorbitol for glycine in SFX. This factor was further confirmed by measuring the gamma dose response of glycine in FX and in SFX for four different doses, viz. 37.8, 75.5, 151 and 302 Gy. It was observed that dose response of glycine in SFX is about 77% more than that of glycine in FX. The maximum variation observed in response of glycine in FX or SFX was found to be within ±1.5%.

Comparative mapping of the wild perennial Glycine latifolia and soybean (G. max reveals extensive chromosome rearrangements in the genus Glycine.

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Sungyul Chang

Full Text Available Soybean (Glycine max L. Mer., like many cultivated crops, has a relatively narrow genetic base and lacks diversity for some economically important traits. Glycine latifolia (Benth. Newell & Hymowitz, one of the 26 perennial wild Glycine species related to soybean in the subgenus Glycine Willd., shows high levels of resistance to multiple soybean pathogens and pests including Alfalfa mosaic virus, Heterodera glycines Ichinohe and Sclerotinia sclerotiorum (Lib. de Bary. However, limited information is available on the genomes of these perennial Glycine species. To generate molecular resources for gene mapping and identification, high-density linkage maps were constructed for G. latifolia using single nucleotide polymorphism (SNP markers generated by genotyping by sequencing and evaluated in an F2 population and confirmed in an F5 population. In each population, greater than 2,300 SNP markers were selected for analysis and segregated to form 20 large linkage groups. Marker orders were similar in the F2 and F5 populations. The relationships between G. latifolia linkage groups and G. max and common bean (Phaseolus vulgaris L. chromosomes were examined by aligning SNP-containing sequences from G. latifolia to the genome sequences of G. max and P. vulgaris. Twelve of the 20 G. latifolia linkage groups were nearly collinear with G. max chromosomes. The remaining eight G. latifolia linkage groups appeared to be products of multiple interchromosomal translocations relative to G. max. Large syntenic blocks also were observed between G. latifolia and P. vulgaris. These experiments are the first to compare genome organizations among annual and perennial Glycine species and common bean. The development of molecular resources for species closely related to G. max provides information into the evolution of genomes within the genus Glycine and tools to identify genes within perennial wild relatives of cultivated soybean that could be beneficial to soybean

A thermodynamic framework for understanding temperature sensing by transient receptor potential (TRP) channels.

Science.gov (United States)

Clapham, David E; Miller, Christopher

2011-12-06

The exceptionally high temperature sensitivity of certain transient receptor potential (TRP) family ion channels is the molecular basis of hot and cold sensation in sensory neurons. The laws of thermodynamics dictate that opening of these specialized TRP channels must involve an unusually large conformational standard-state enthalpy, ΔH(o): positive ΔH(o) for heat-activated and negative ΔH(o) for cold-activated TRPs. However, the molecular source of such high-enthalpy changes has eluded neurobiologists and biophysicists. Here we offer a general, unifying mechanism for both hot and cold activation that recalls long-appreciated principles of protein folding. We suggest that TRP channel gating is accompanied by large changes in molar heat capacity, ΔC(P). This postulate, along with the laws of thermodynamics and independent of mechanistic detail, leads to the conclusion that hot- and cold-sensing TRPs operate by identical conformational changes.

MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells.

Science.gov (United States)

Karasawa, Jun-ichi; Yamamoto, Hideko; Yamamoto, Toshifumi; Sagi, Naoki; Horikomi, Kazutoshi; Sora, Ichiro

2002-02-22

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.

A glycine insertion in the estrogen-related receptor (ERR is associated with enhanced expression of three cytochrome P450 genes in transgenic Drosophila melanogaster.

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Weilin Sun

Full Text Available Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4'-dichlorodiphenyltrichloroethane (DDT resistant strains the glucocorticoid receptor-like (GR-like potential transcription factor binding motifs (TFBMs have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR in D. melanogaster is an estrogen-related receptor (ERR gene, which has two predicted alternative splice isoforms (ERRa and ERRb. Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G. Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera.

The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.

OpenAIRE

Trost, C; Bergs, C; Himmerkus, N; Flockerzi, V

2001-01-01

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1...

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury

OpenAIRE

Zimmer, M. Beth; Goshgarian, Harry G.

2006-01-01

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague Dawley rats were hemisected at the C2 spinal cord level. Fo...

Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

Science.gov (United States)

Therien, J P Daniel; Baenziger, John E

2017-03-27

Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.

TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

Science.gov (United States)

Ortíz-Rentería, Miguel; Juárez-Contreras, Rebeca; González-Ramírez, Ricardo; Islas, León D; Sierra-Ramírez, Félix; Llorente, Itzel; Simon, Sidney A; Hiriart, Marcia; Rosenbaum, Tamara; Morales-Lázaro, Sara L

2018-02-13

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Glycine and a glycine dehydrogenase (GLDC) SNP as citalopram/escitalopram response biomarkers in depression: pharmacometabolomics-informed pharmacogenomics.

Science.gov (United States)

Ji, Y; Hebbring, S; Zhu, H; Jenkins, G D; Biernacka, J; Snyder, K; Drews, M; Fiehn, O; Zeng, Z; Schaid, D; Mrazek, D A; Kaddurah-Daouk, R; Weinshilboum, R M

2011-01-01

Major depressive disorder (MDD) is a common psychiatric disease. Selective serotonin reuptake inhibitors (SSRIs) are an important class of drugs used in the treatment of MDD. However, many patients do not respond adequately to SSRI therapy. We used a pharmacometabolomics-informed pharmacogenomic research strategy to identify citalopram/escitalopram treatment outcome biomarkers. Metabolomic assay of plasma samples from 20 escitalopram remitters and 20 nonremitters showed that glycine was negatively associated with treatment outcome (P = 0.0054). This observation was pursued by genotyping tag single-nucleotide polymorphisms (SNPs) for genes encoding glycine synthesis and degradation enzymes, using 529 DNA samples from SSRI-treated MDD patients. The rs10975641 SNP in the glycine dehydrogenase (GLDC) gene was associated with treatment outcome phenotypes. Genotyping for rs10975641 was carried out in 1,245 MDD patients in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, and its presence was significant (P = 0.02) in DNA taken from these patients. These results highlight a possible role for glycine in SSRI response and illustrate the use of pharmacometabolomics to "inform" pharmacogenomics.

Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission

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Sorana Ciura

2018-05-01

Full Text Available Summary: The maintenance of hydromineral homeostasis requires bidirectional detection of changes in extracellular fluid osmolality by primary osmosensory neurons (ONs in the organum vasculosum laminae terminalis (OVLT. Hypertonicity excites ONs in part through the mechanical activation of a variant transient receptor potential vanilloid-1 channel (dn-Trpv1. However, the mechanism by which local hypotonicity inhibits ONs in the OVLT remains unknown. Here, we show that hypotonicity can reduce the basal activity of dn-Trpv1 channels and hyperpolarize acutely isolated ONs. Surprisingly, we found that mice lacking dn-Trpv1 maintain normal inhibitory responses to hypotonicity when tested in situ. In the intact setting, hypotonicity inhibits ONs through a non-cell-autonomous mechanism that involves glial release of the glycine receptor agonist taurine through hypotonicity activated anion channels (HAAC that are activated subsequent to Ca2+ influx through Trpv4 channels. Our study clarifies how Trpv4 channels contribute to the inhibition of OVLT ONs during hypotonicity in situ. : Ciura et al. show that osmosensory neurons in organum vasculosum lamina terminalis are inhibited by hypotonicity. This effect is triggered by activation of Trpv4 channels and Ca2+ accumulation in astrocytes, causing these cells to release taurine through anion channels. Taurine inhibits firing by activating glycine receptors on the osmosensory neurons. Keywords: hyptonicity, taurine, TRPV, osmosensitive, gliotransmission, swelling

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

Science.gov (United States)

Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

2014-09-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

The Proteoglycan Syndecan 4 Regulates Transient Receptor Potential Canonical 6 Channels via RhoA/ROCK Signaling

DEFF Research Database (Denmark)

Liu, Ying; Echtermeyer, Frank; Thilo, Florian

2012-01-01

OBJECTIVE: Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular perme...... permeability, in a RhoA/ROCK-dependent manner. METHODS AND RESULTS: Sdc4 knockout (Sdc4(-/-)) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P...

Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

International Nuclear Information System (INIS)

Curtis, B.M.

1986-01-01

Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [ 3 H]nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed α(Mr 135,000), β(Mr 50,000), and γ(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The α and γ subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the α, β, and γ subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

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Laurent Mackay

2017-07-01

Full Text Available The P2X4 receptor (P2X4R is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM, a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia.

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J Pedro Fernández-Murray

2016-01-01

Full Text Available Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.

Citral Sensing by TRANSient Receptor Potential Channels in Dorsal Root Ganglion Neurons

Science.gov (United States)

Stotz, Stephanie C.; Vriens, Joris; Martyn, Derek; Clardy, Jon; Clapham, David E.

2008-01-01

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1–3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin. PMID:18461159

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

Science.gov (United States)

Gregorio-Teruel, Lucia; Valente, Pierluigi; González-Ros, José Manuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2014-03-01

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception.

Science.gov (United States)

Mancuso, Giuseppe; Borgonovo, Gigliola; Scaglioni, Leonardo; Bassoli, Angela

2015-10-16

Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

The light activated alkylation of glycine

International Nuclear Information System (INIS)

Knowles, H.S.

2001-04-01

The work contained in this thesis focuses on the light-initiated alkylation of the α-centre of glycine compounds. The elaboration of the glycines in this manner represents a versatile, clean and cost effective alternative to ionic routes to higher α-amino acids. Preliminary investigations demonstrated that a range of nitrogen protecting groups were compatible with the radical alkylation. A variety of solvents could also be used although solvents with easily removable hydrogen atoms were found to interfere with the alkylation. Furthermore, a number of photo-initiators were investigated and the use of di-tert-butyl peroxide was found to afford the desired phenylalanine products in up to 27% yield (54% based on recovered starting material) when toluene was used as the alkylating agent. A range of different precursor concentrations was investigated and it was found that the optimum concentration of the glycine precursor was 0.13 mol dm -3 ; the phenylalanine yields were reduced when the concentration was less than this value. Owing to the poor UV absorption by di-tert-butyl peroxide, benzophenone (an effective photosensitiser) was added to the reaction mixture and this was shown to increase the alkylation yields. The ratio of reagents which produced the highest yield of phenylalanine products was found to be 1 : 5 : 5 : 10 for glycine : di-tert-butyl peroxide : benzophenone : toluene. This produced the phenylalanine product in up to 37% yield (57% based on recovered starting material). A number of substituents. (e.g. F, Cl etc.) could be attached to the aromatic ring of the toluene alkylating agent, affording substituted phenylalanines in 5 - 36% under these conditions. The formation of chiral phenylalanine products was probed by reacting glycine precursors bearing chiral auxiliaries. However, low diastereoselectivities were observed; the d.r. ranged from 1 : 1.1 to 1 : 1.5 only when chiral ester and amide protecting groups were used. In the final chapter, the �

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

International Nuclear Information System (INIS)

McCue, Jeffrey M.; Driscoll, William J.; Mueller, Gregory P.

2008-01-01

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo

Mutation of a zinc-binding residue in the glycine receptor α1 subunit changes ethanol sensitivity in vitro and alcohol consumption in vivo.

Science.gov (United States)

McCracken, Lindsay M; Blednov, Yuri A; Trudell, James R; Benavidez, Jillian M; Betz, Heinrich; Harris, R Adron

2013-02-01

Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs), which are potentiated by millimolar concentrations of ethanol. In addition, zinc ions also modulate GlyR function, and recent evidence suggests that physiologic concentrations of zinc enhance ethanol potentiation of GlyRs. Here, we first built a homology model of a zinc-bound GlyR using the D80 position as a coordination site for a zinc ion. Next, we investigated in vitro the effects of zinc on ethanol action at recombinant wild-type (WT) and mutant α1 GlyRs containing the D80A substitution, which eliminates zinc potentiation. At D80A GlyRs, the effects of 50 and 200 mM ethanol were reduced as compared with WT receptors. Also, in contrast to what was seen with WT GlyRs, neither adding nor chelating zinc changed the magnitude of ethanol enhancement of mutant D80A receptors. Next, we evaluated the in vivo effects of the D80A substitution by using heterozygous Glra1(D80A) knock-in (KI) mice. The KI mice showed decreased ethanol consumption and preference, and they displayed increased startle responses compared with their WT littermates. Other behavioral tests, including ethanol-induced motor incoordination and strychnine-induced convulsions, revealed no differences between the KI and WT mice. Together, our findings indicate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc binding at the D80 position may be important for mediating some of the behavioral effects of ethanol action at GlyRs.

GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

Science.gov (United States)

Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

Science.gov (United States)

Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

2013-03-01

Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

Science.gov (United States)

Kim, Young-Hwan; Jeong, Ji-Hyun; Ahn, Duck-Sun; Chung, Seungsoo

2016-08-26

Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension. Copyright © 2016 Elsevier Inc. All rights reserved.

Glycine-U-14C metabolism in young rats fed the 10% casein diets containing excess glycine

International Nuclear Information System (INIS)

Takeuchi, Hisanao; Wakatsuki, Tetsuo; Muramatsu, Keiichiro

1975-01-01

Nine hours after rats fed ad libitum for 14 days a 10% casein diet (10C), a 10% casein diet containing 7% glycine (10C7G) and a 10% casein diet containing 7% glycine with 1.4% L-arginine.HCl and 0.9% L-methionine (10C7GArgMet) were force-fed 10 ml of each diet suspension containing 5μCi of glycine-U- 14 C per 100 g of body weight, the radioactivity recoveries of 14 C in expired CO 2 , tissue components and urine were determined. The radioactivity recovery of 14 C in the expired CO 2 of the 10C7G group was generally higher than that of the 10C7GArgMet group. The recovery of 14 C in the trichloroacetic acid (TCA) soluble fraction of muscle of the 10C7G and the 10C7GArgMet groups were greater than that of the 10C group. The recoveries of 14 C in the TCA soluble fraction and protein of plasma and liver, and the muscle protein were negligible in all the groups. The amount of glycine- 14 C incorporated into the carcass lipids of the 10C7GArgMet group was larger than that of other groups. The recoveries of 14 C in the liver and muscle glycogen, and liver lipids were remarkably small in all the groups. From the above results, it was suggested that the degradation of glycine- 14 C to expiratory CO 2 was not accelerated, but the rate of incorporation of the isotope into carcass lipids was increased by the supplementation of L-arginine and L-methionine to the 10C7G diet as compared with that of rats fed the 10C7G diet. (JPN)

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

Science.gov (United States)

Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

2005-01-01

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception

Directory of Open Access Journals (Sweden)

Giuseppe Mancuso

2015-10-01

Full Text Available Ruta graveolens (rue is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists

DEFF Research Database (Denmark)

Pless, Stephan Alexander; Hanek, Ariele P; Price, Kerry L

2011-01-01

. In the current study, we investigated whether the lower efficacy agonists of the human GlyR β-alanine and taurine also form cation-π interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-π interactions between Phe159 and the amino groups of β-alanine and taurine....... The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that β-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have...... similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of β-alanine and taurine....

GLYX-13, a NMDA receptor glycine-site functional partial agonist, induces antidepressant-like effects without ketamine-like side effects.

Science.gov (United States)

Burgdorf, Jeffrey; Zhang, Xiao-lei; Nicholson, Katherine L; Balster, Robert L; Leander, J David; Stanton, Patric K; Gross, Amanda L; Kroes, Roger A; Moskal, Joseph R

2013-04-01

Recent human clinical studies with the NMDA receptor (NMDAR) antagonist ketamine have revealed profound and long-lasting antidepressant effects with rapid onset in several clinical trials, but antidepressant effects were preceded by dissociative side effects. Here we show that GLYX-13, a novel NMDAR glycine-site functional partial agonist, produces an antidepressant-like effect in the Porsolt, novelty induced hypophagia, and learned helplessness tests in rats without exhibiting substance abuse-related, gating, and sedative side effects of ketamine in the drug discrimination, conditioned place preference, pre-pulse inhibition and open-field tests. Like ketamine, the GLYX-13-induced antidepressant-like effects required AMPA/kainate receptor activation, as evidenced by the ability of NBQX to abolish the antidepressant-like effect. Both GLYX-13 and ketamine persistently (24 h) enhanced the induction of long-term potentiation of synaptic transmission and the magnitude of NMDAR-NR2B conductance at rat Schaffer collateral-CA1 synapses in vitro. Cell surface biotinylation studies showed that both GLYX-13 and ketamine led to increases in both NR2B and GluR1 protein levels, as measured by Western analysis, whereas no changes were seen in mRNA expression (microarray and qRT-PCR). GLYX-13, unlike ketamine, produced its antidepressant-like effect when injected directly into the medial prefrontal cortex (MPFC). These results suggest that GLYX-13 produces an antidepressant-like effect without the side effects seen with ketamine at least in part by directly modulating NR2B-containing NMDARs in the MPFC. Furthermore, the enhancement of 'metaplasticity' by both GLYX-13 and ketamine may help explain the long-lasting antidepressant effects of these NMDAR modulators. GLYX-13 is currently in a Phase II clinical development program for treatment-resistant depression.

Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons.

Science.gov (United States)

Stotz, Stephanie C; Vriens, Joris; Martyn, Derek; Clardy, Jon; Clapham, David E

2008-05-07

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.

Role of aryl hydrocarbon receptor nuclear translocator in KATP channel-mediated insulin secretion in INS-1 insulinoma cells

International Nuclear Information System (INIS)

Kim, Ji-Seon; Zheng Haifeng; Kim, Sung Joon; Park, Jong-Wan; Park, Kyong Soo; Ho, Won-Kyung; Chun, Yang-Sook

2009-01-01

Aryl hydrocarbon receptor nuclear translocator (ARNT) has been known to participate in cellular responses to xenobiotic and hypoxic stresses, as a common partner of aryl hydrocarbon receptor and hypoxia inducible factor-1/2α. Recently, it was reported that ARNT is essential for adequate insulin secretion in response to glucose input and that its expression is downregulated in the pancreatic islets of diabetic patients. In the present study, the authors addressed the mechanism by which ARNT regulates insulin secretion in the INS-1 insulinoma cell line. In ARNT knock-down cells, basal insulin release was elevated, but insulin secretion was not further stimulated by a high-glucose challenge. Electrophysiological analyses revealed that glucose-dependent membrane depolarization was impaired in these cells. Furthermore, K ATP channel activity and expression were reduced. Of two K ATP channel subunits, Kir6.2 was found to be positively regulated by ARNT at the mRNA and protein levels. Based on these results, the authors suggest that ARNT expresses K ATP channel and by so doing regulates glucose-dependent insulin secretion.

Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons.

Science.gov (United States)

Zhang, L; Kolaj, M; Renaud, L P

2015-12-17

postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

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Rene Barro-Soria

Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

Gas-phase reactions of glycine, alanine, valine and their N-methyl derivatives with the nitrosonium ion, NO+.

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Freitas, M A; O'Hair, R A; Schmidt, J A; Tichy, S E; Plashko, B E; Williams, T D

1996-10-01

The gas-phase reactions of the nitrosonium ion, NO+ with the amino acids glycine, alanine and valine and their N-methyl derivatives were investigated under chemical ionization mass spectrometric (CIMS) conditions. Two products were observed in all cases: the formation of the iminium ion and the formation of an [M-H]+ ion. The latter product is consistent with a reaction channel involving hydride abstraction by NO+, and was confirmed by (i) examining the Ar+CI mass spectra of the same amino acids under similar source conditions and (ii) examining the unimolecular fragmentation reactions of the [M + H]+ ions of the N-nitroso-N-methyl derivatives of each of the amino acids in a tandem mass spectrometer. Further insights into the reaction of glycine with NO+ were obtained by performing ab initio calculations (at the MP2/6-31G* parallel HF/6-31G* level). These results indicate that four reactions are thermodynamically viable for glycine: (i) hydride abstraction; (ii) iminium ion formation (with concomitant loss of HONO and CO); (iii) diazonium ion formation; and (iv) diazonium ion formation followed by loss of N2. Possible reasons why reactions (iii) and (iv) are not observed are discussed, and comparisons with solution reactivity and the gas-phase reactivity of NO+ are also made.

Fast heavy-ion radiation damage of glycine in aqueous solution

Energy Technology Data Exchange (ETDEWEB)

Nomura, Shinji [Department of Nuclear Engineering, Kyoto University, Kyoto 615-8530 (Japan); Tsuchida, Hidetsugu, E-mail: tsuchida@nucleng.kyoto-u.ac.jp [Department of Nuclear Engineering, Kyoto University, Kyoto 615-8530 (Japan); Quantum Science and Engineering Center, Kyoto University, Uji 611-0011 (Japan); Furuya, Ryosuke [Department of Nuclear Engineering, Kyoto University, Kyoto 615-8530 (Japan); Majima, Takuya; Itoh, Akio [Department of Nuclear Engineering, Kyoto University, Kyoto 615-8530 (Japan); Quantum Science and Engineering Center, Kyoto University, Uji 611-0011 (Japan)

2016-12-15

Fast heavy-ion radiolysis of biomolecules in aqueous solution is investigated for an atomistic understanding of radiation damage to normal cells during heavy-particle beam therapy. The smallest amino acid glycine was used as a model biomaterial. Microjets of aqueous glycine solutions under vacuum were irradiated with 4.0-MeV carbon ions corresponding to energies in the Bragg peak region. To understand the effects of the water environment on molecular damage, the yield of glycine dissociation was measured by secondary ion mass spectroscopy. The yield was significantly reduced relative to gas-phase glycine targets. This implies that the numerous water molecules surrounding a single glycine molecule act as a buffer that suppresses dissociation. This is an environmental effect similar to that observed for other biomolecular cluster targets.

Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats.

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Kubo, Takuro; Tsuji, Shunichiro; Amano, Tsukuru; Yoshino, Fumi; Niwa, Yoko; Kasahara, Kyoko; Yoshida, Saori; Mukaisho, Ken-Ichi; Sugihara, Hiroyuki; Tanaka, Sachiko; Kimura, Fuminori; Takahashi, Kentaro; Murakami, Takashi

2017-10-30

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is associated with sensitivity to cold sensation in mammals. A previous study demonstrated that TRPM8 was overexpressed in the skin of ovariectomized (OVX) rats due to the loss of estrogen. In the present study, we investigated whether estrogen replacement restricts overexpression of the TRPM8 channel in the skin of OVX rats. We divided 15 Sprague Dawley rats into three groups: a non-operated group (NON-OPE), an ovariectomy group (OVX), and a group subjected to estrogen replacement during 4 weeks beginning 7 days after ovariectomy (OVX + E2). Five weeks later, TRPM8 channel mRNA and protein in lumbar skin were quantified by real-time RT-PCR, protein ELISA, and immunohistochemistry. The OVX + E2 group exhibited a trend for decreased expression of the TRPM8 channel in the lumbar skin in comparison with the OVX group, whereas ELISA data and immunohistochemistry data and immunohistochemistry graphs relating to TRPM8 protein did not show any obvious differences between the OVX group and the OVX + E2 group. Estrogen replacement may restrict the overexpression of TRPM8 in the dermis of OVX rats.

Electrophysiological evidence of increased glycine receptor-mediated phasic and tonic inhibition by blockade of glycine transporters in spinal superficial dorsal horn neurons of adult mice

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Misa Oyama

2017-03-01

Full Text Available To understand the synaptic and/or extrasynaptic mechanisms underlying pain relief by blockade of glycine transporter subtypes GlyT1 and GlyT2, whole-cell recordings were made from dorsal horn neurons in spinal slices from adult mice, and the effects of NFPS and ALX-1393, selective GlyT1 and GlyT2 inhibitors, respectively, on phasic evoked or miniature glycinergic inhibitory postsynaptic currents (eIPSCs or mIPSCs were examined. NFPS and ALX-1393 prolonged the decay phase of eIPSCs without affecting their amplitude. In the presence of tetrodotoxin to record mIPSCs, NFPS and ALX-1393 induced a tonic inward current that was reversed by strychnine. Although NFPS had no statistically significant influences on mIPSCs, ALX-1393 significantly increased their frequency. We then further explored the role of GlyTs in the maintenance of glycinergic IPSCs. To facilitate vesicular release of glycine, repetitive high-frequency stimulation (HFS was applied at 10 Hz for 3 min during continuous recordings of eIPSCs at 0.1 Hz. Prominent suppression of eIPSCs was evident after HFS in the presence of ALX-1393, but not NFPS. Thus, it appears that phasic and tonic inhibition may contribute to the analgesic effects of GlyT inhibitors. However, reduced glycinergic inhibition due to impaired vesicular refilling could hamper the analgesic efficacy of GlyT2 inhibitors.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

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Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons.

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Stephanie C Stotz

2008-05-01

Full Text Available Transient receptor potential (TRP ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1, and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate, consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.

Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

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Dicke, J M; Verges, D; Kelley, L K; Smith, C H

1993-01-01

Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

Ryanodine receptors, voltage-gated calcium channels and their relationship with protein kinase A in the myocardium

Czech Academy of Sciences Publication Activity Database

Petrovič, Miloš; Valeš, Karel; Putnikovič, B.; Djulejič, V.; Mitrovič, D. M.

2008-01-01

Roč. 57, č. 2 (2008), s. 141-149 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA309/07/0271; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : rynodine receptors * kalcium channels Subject RIV: ED - Physiology Impact factor: 1.653, year: 2008

Functional transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 channels along different segments of the renal vasculature

DEFF Research Database (Denmark)

Chen, L; Kaßmann, M; Sendeski, M

2015-01-01

with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.......AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized...... that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels. METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels. RESULTS: The TRPV1 agonist capsaicin relaxed mouse...

The glycine reuptake inhibitor org 25935 interacts with basal and ethanol-induced dopamine release in rat nucleus accumbens.

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Lidö, Helga Höifödt; Stomberg, Rosita; Fagerberg, Anne; Ericson, Mia; Söderpalm, Bo

2009-07-01

The mesolimbic dopamine (DA) projection from the ventral tegmental area to nucleus accumbens (nAc), a central part of the reward system, is activated by ethanol (EtOH) and other drugs of abuse. We have previously demonstrated that the glycine receptor in the nAc and its amino acid agonists may be implicated in the DA activation and reinforcing properties of EtOH. We have also reported that the glycine transporter 1 inhibitor, Org 25935, produces a robust and dose-dependent decrease in EtOH consumption in Wistar rats. The present study explores the interaction between EtOH and Org 25935 with respect to DA levels in the rat nAc. The effects of Org 25935 (6 mg/kg, i.p.) and/or EtOH (2.5 g/kg, i.p.) on accumbal DA levels were examined by means of in vivo microdialysis (coupled to HPLC-ED) in freely moving male Wistar rats. The effect of Org 25935 on accumbal glycine output was also investigated. Systemic Org 25935 increased DA output in a subpopulation of rats (52% in Experiment 1 and 38% in Experiment 2). In Experiment 2, EtOH produced a significant increase in DA levels in vehicles (35%) and in Org 25935 nonresponders (19%), whereas EtOH did not further increase the DA level in rats responding to Org 25935 (2%). The same dose of Org 25935 increased glycine levels by 87% in nAc. This study demonstrates that Org 25935, probably via increased glycine levels, (i) counteracts EtOH-induced increases of accumbal DA levels and (ii) increases basal DA levels in a subpopulation of rats. The results are in line with previous findings and it is suggested that the effects observed involve interference with accumbal GlyRs and are related to the alcohol consumption modulating effect of Org 25935.

Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes

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Pedro L. Flores

2017-06-01

Full Text Available Maitotoxin (MTX is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP. Several reports indicate that MTX is an activator of non-selective cation channels (NSCC in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA approach and the two-electrode voltage-clamp technique (TEVC. The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1 protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

Production of 1-carbon units from glycine is extensive in healthy men and women.

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Lamers, Yvonne; Williamson, Jerry; Theriaque, Douglas W; Shuster, Jonathan J; Gilbert, Lesa R; Keeling, Christine; Stacpoole, Peter W; Gregory, Jesse F

2009-04-01

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO(2), NH(3), and a 1-carbon unit. CO(2) also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in CO(2) generation in healthy volunteers (3 male, 3 female, aged 21-26 y), primed, constant infusions were conducted using 9.26 micromol x h(-1) x kg(-1) of [1,2-(13)C]glycine and 1.87 micromol x h(-1) x kg(-1) of [5,5,5-(2)H(3)]leucine, followed by an infusion protocol using [1-(13)C]glycine as the glycine tracer. The time period between the infusion protocols was >6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-(13)C]glycine and [5,5,5-(2)H(3)]leucine and with [1-(13)C]glycine and [5,5,5-(2)H(3)]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-(13)C]glycine tracer, breath CO(2) data showed a total rate of glycine decarboxylation of 96 +/- 8 micromol x h(-1) x kg(-1), which was 22 +/- 3% of whole-body glycine flux. In contrast, infusion of [1,2-(13)C]glycine yielded a glycine-to-CO(2) flux of 146 +/- 37 micromol x h(-1) x kg(-1) (P = 0.026). By difference, this implies a rate of CO(2) formation from the glycine 2-carbon of 51 +/- 40 micromol x h(-1) x kg(-1), which accounts for approximately 35% of the total CO(2) generated in glycine catabolism. These findings also indicate that approximately 65% of the CO(2) generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate ( approximately 96 micromol x h(-1) x kg(-1)), which is approximately 20 times the demand for methyl groups for homocysteine remethylation.

Paving the way to understand humoral autoantibody epilepsy on the molecular level

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Guiscard eSeebohm

2015-07-01

Full Text Available Correct function of neuronal networks is enabled by a delicate interplay among neurons communicating with each other. One of the keys is the communication at chemical synapses where neurotransmitters like glutamate, GABA and glycine enable signal transfer over the synaptic cleft. Thereby, the neurotransmitters are released from the presynapse and bind as ligands to specific receptors at the postsynaptic side to allow for modulation of the postsynaptic membrane potentials. The postsynaptic electrical signal, which is highly modulated by voltage gated ion channels, spreads over the dendritic tree and is thus integrated to allow for generation of action potentials at the axon hillock. This concert of receptors and voltage gated ion channels depends on correct function of all its components. Misfunction of receptors and/or voltage-gated potassium channels (VGKC leads to diverse adverse effects in patients. Such malfunctions can be the result of inherited genetic alterations or pharmacological side effects by drugs. Recently, auto-antibodies targeting receptor or channel complexes like NMDAR, AMPAR, GABA-receptors, glycine-receptors, LGI1 or CASPR2 (previously termed VGKC-complex antibodies have been discovered. The presence of specific auto-antibodies against these targets associates with severe forms of antibody-mediated encephalitis. Understanding the molecular details of auto-antibody actions on receptor and VGKC complexes is highly desirable and may open the path to develop specific therapies to treat humoral autoimmune encephalitis. Here, we summarize the current knowledge and discuss technical approaches to fill the gap of knowledge. These techniques include electrophysiology, biochemical approaches for epitope mapping and in silico modeling to simulate molecular interactions between autoantibody and its molecular target.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models

Czech Academy of Sciences Publication Activity Database

Mackay, L.; Zemková, Hana; Stojilkovic, S. S.; Sherman, A.; Khadra, A.

2017-01-01

Roč. 13, č. 7 (2017), č. článku e1005643. ISSN 1553-734X R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : Markov models * ion channel gating * sensory receptors * cation s Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.542, year: 2016

Temperature and Voltage Coupling to Channel Opening in Transient Receptor Potential Melastatin 8 (TRPM8)*♦

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Raddatz, Natalia; Castillo, Juan P.; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

2014-01-01

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol−1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

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Lin, Yu-Ping; Nelson, Charmaine; Kramer, Holger; Parekh, Anant B

2018-04-19

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca 2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca 2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca 2+ channels and suggest a therapeutic strategy for treating mite-induced asthma. Copyright © 2018 Elsevier Inc. All rights reserved.

Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

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Paula L Diaz-Sylvester

Full Text Available Cardiac ryanodine receptor (RyR2 function is modulated by Ca(2+ and Mg(2+. To better characterize Ca(2+ and Mg(2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+: Mg(2+, Ca(2+, Sr(2+, Ba(2+ were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+ or Sr(2+. This activation was interfered by Mg(2+ and Ba(2+ acting at low affinity M(2+-unspecific binding sites. When testing the effects of luminal M(2+ as current carriers, all M(2+ increased maximal RyR2 open probability (compared to Cs(+, suggesting the existence of low affinity activating M(2+-unspecific sites at the luminal surface. Responses to M(2+ vary from channel to channel (heterogeneity. However, with luminal Ba(2+or Mg(2+, RyR2 were less sensitive to cytosolic Ca(2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+or Sr(2+. Kinetics of RyR2 with mixtures of luminal Ba(2+/Ca(2+ and additive action of luminal plus cytosolic Ba(2+ or Mg(2+ suggest luminal M(2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+/Sr(2+-specific sites, which stabilize high P(o mode (less voltage-dependent and increase RyR2 sensitivity to cytosolic Ca(2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+ binding sites (specific for Ca(2+ and unspecific for Ca(2+/Mg(2+ that dynamically modulate channel activity and gating status, depending on SR voltage.

Histamine facilitates GABAergic transmission in the rat entorhinal cortex: Roles of H1 and H2 receptors, Na+ -permeable cation channels, and inward rectifier K+ channels.

Science.gov (United States)

Cilz, Nicholas I; Lei, Saobo

2017-05-01

In the brain, histamine (HA) serves as a neuromodulator and a neurotransmitter released from the tuberomammillary nucleus (TMN). HA is involved in wakefulness, thermoregulation, energy homeostasis, nociception, and learning and memory. The medial entorhinal cortex (MEC) receives inputs from the TMN and expresses HA receptors (H 1 , H 2 , and H 3 ). We investigated the effects of HA on GABAergic transmission in the MEC and found that HA significantly increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with an EC 50 of 1.3 µM, but failed to significantly alter sIPSC amplitude. HA-induced increases in sIPSC frequency were sensitive to tetrodotoxin (TTX), required extracellular Ca 2+ , and persisted when GDP-β-S, a G-protein inactivator, was applied postsynaptically via the recording pipettes, indicating that HA increased GABA release by facilitating the excitability of GABAergic interneurons in the MEC. Recordings from local MEC interneurons revealed that HA significantly increased their excitability as determined by membrane depolarization, generation of an inward current at -65 mV, and augmentation of action potential firing frequency. Both H 1 and H 2 receptors were involved in HA-induced increases in sIPSCs and interneuron excitability. Immunohistochemical staining showed that both H 1 and H 2 receptors are expressed on GABAergic interneurons in the MEC. HA-induced depolarization of interneurons involved a mixed ionic mechanism including activation of a Na + -permeable cation channel and inhibition of a cesium-sensitive inward rectifier K + channel, although HA also inhibited the delayed rectifier K + channels. Our results may provide a cellular mechanism, at least partially, to explain the roles of HA in the brain. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system.

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Zimmermann, Katharina; Lennerz, Jochen K; Hein, Alexander; Link, Andrea S; Kaczmarek, J Stefan; Delling, Markus; Uysal, Serdar; Pfeifer, John D; Riccio, Antonio; Clapham, David E

2011-11-01

Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

Genetics Home Reference: glycine encephalopathy

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... seizures. As they get older, many develop intellectual disability, abnormal movements, and behavioral problems. Other atypical types of glycine encephalopathy appear later in childhood or adulthood ...

First-principles study of the formation of glycine-producing radicals from common interstellar species

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Sato, Akimasa; Kitazawa, Yuya; Ochi, Toshiro; Shoji, Mitsuo; Komatsu, Yu; Kayanuma, Megumi; Aikawa, Yuri; Umemura, Masayuki; Shigeta, Yasuteru

2018-03-01

Glycine, the simplest amino acid, has been intensively searched for in molecular clouds, and the comprehensive clarification of the formation path of interstellar glycine is now imperative. Among all the possible glycine formation pathways, we focused on the radical pathways revealed by Garrod (2013). In the present study, we have precisely investigated all the chemical reaction steps related to the glycine formation processes based on state-of-the-art density functional theory (DFT) calculations. We found that two reaction pathways require small activation barriers (ΔE‡ ≤ 7.75 kJ mol-1), which demonstrates the possibility of glycine formation even at low temperatures in interstellar space if the radical species are generated. The origin of carbon and nitrogen in the glycine backbone and their combination patterns are further discussed in relation to the formation mechanisms. According to the clarification of the atomic correspondence between glycine and its potential parental molecules, it is shown that the nitrogen and two carbons in the glycine can originate in three common interstellar molecules, methanol, hydrogen cyanide, and ammonia, and that the source molecules of glycine can be described by any of their combinations. The glycine formation processes can be categorized into six patterns. Finally, we discussed two other glycine formation pathways expected from the present DFT calculation results.

Growth and antimicrobial studies of γ-glycine crystal grown using CuSO4

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Vijayalakshmi, V.; Dhanasekaran, P.

2018-05-01

In the current work single crystals of pure and 1M of CuSO4-added glycine were grown by slow evaporation method and its optical and antimicrobial properties were studied. The Polymorph of glycine transforms from a-glycine to γ-glycine due to the incorporation of CuSO4 on glycine was affirmed by the PXRD and FTIR studies. The impact of CuSO4 on the antimicrobial action of the grown samples was deliberate by utilizing the agar diffusion method.

Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1

Czech Academy of Sciences Publication Activity Database

Toušová, Karolina; Sušánková, Klára; Teisinger, Jan; Vyklický st., Ladislav; Vlachová, Viktorie

2004-01-01

Roč. 47, č. 2 (2004), s. 273-285 ISSN 0028-3908 R&D Projects: GA ČR GA305/03/0802; GA ČR GA309/02/1479; GA MŠk LN00B122 Institutional research plan: CEZ:AV0Z5011922 Keywords : vanilloid receptor * TRP channels * capsaicin Subject RIV: ED - Physiology Impact factor: 3.734, year: 2004

A combined role of calcium channel blockers and angiotensin receptor blockers in stroke prevention

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Ji-Guang Wang

2009-07-01

Full Text Available Ji-Guang WangCentre for Epidemiological Studies and Clinical Trials, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaAbstract: Stroke is a leading cause of death and disability worldwide. The importance of lowering blood pressure for reducing the risk of stroke is well established. However, not all the benefits of antihypertensive treatments in stroke can be accounted for by reductions in BP and there may be differences between antihypertensive classes as to which provides optimal protection. Dihydropyridine calcium channel blockers, such as amlodipine, and angiotensin receptor blockers, such as valsartan, represent the two antihypertensive drug classes with the strongest supportive data for the prevention of stroke. Therefore, when combination therapy is required, a combination of these two antihypertensive classes represents a logical approach.Keywords: stroke, angiotensin, calcium channel, cerebrovascular, hypertension, blood pressure

Duplicated Gephyrin Genes Showing Distinct Tissue Distribution and Alternative Splicing Patterns Mediate Molybdenum Cofactor Biosynthesis, Glycine Receptor Clustering, and Escape Behavior in Zebrafish*

Science.gov (United States)

Ogino, Kazutoyo; Ramsden, Sarah L.; Keib, Natalie; Schwarz, Günter; Harvey, Robert J.; Hirata, Hiromi

2011-01-01

Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABAA receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish. PMID:20843816

Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism.

Science.gov (United States)

Rossokhin, Alexey V; Sharonova, Irina N; Bukanova, Julia V; Kolbaev, Sergey N; Skrebitsky, Vladimir G

2014-11-01

GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of β-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block. Copyright © 2014 Elsevier Inc. All rights reserved.

Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

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Pallavi A Bafna

2008-06-01

Full Text Available Gating of nicotinic acetylcholine receptors from a C(losed to an O(pen conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where CO gating motions generate a lowhigh change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'. This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268 appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.

A weak link in metabolism: the metabolic capacity for glycine ...

Indian Academy of Sciences (India)

Prakash

2009-12-03

Dec 3, 2009 ... glyoxylate, threonine and trimethyllysine (carnitine synthesis), most of them ...... Participation of glycine in porphyrin biosynthesis. Eight glycine ...... normal and streptozotocin-induced diabetic rats; J. Dent. Res. 63 23–27.

Enhanced attachment and growth of periodontal cells on glycine-arginine-glycine-aspartic modified chitosan membranes

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Hsiao-Pei Tu

2016-01-01

Full Text Available Background: Chitosan, a polymeric carbohydrate derived from the exoskeleton of arthropod, has been suggested to be an excellent biomaterial for improving wound healing, especially for bones. To improve the periodontal cell attachment and growth, the cell adhesive peptide glycine-arginine-glycine-aspartic acid (Gly-Arg-Gly-Asp, GRGD grafted chitosan membrane was introduced in this study. Materials and Methods: Two types of commercial chitosan, three types of primary cultured cells, and two established cell lines were used. Human gingival and periodontal fibroblasts (hGF and hPDL, human root derived cell (hRDC, and rat calvaria bone cell (rCalB were cultured on the GRGD-fixed by ultraviolet light photochemical method on the chitosan membrane. With (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assay and propidium iodine (PI staining, the cell adhesion and growth on GRGD-grafted chitosan were examined. Basal mRNA expressions of the receptors for GRGD, integrin αv (ITG αv and ITG β3, in the human gingival fibroblast cell line and mouse osteoblast cell line (MC3T3-E1 were examined with real-time polymerase chain reaction. Results: Because the cell adhesion/growth patterns on two chitosan membranes were similar, the GRGD modification was performed on one membrane (Primex only. For periodontal cells (hGFs, hPDLs, and hRDCs, the number of attached cells were increased on the membrane with the high concentration of GRGD than those on the membrane unmodified or modified with low concentration GRGD. For rCalBs cells, a different pattern was noted: GRGD modification did not enhance the calvaria cells attachment or growth. Moreover, mRNA expressions of ITG αv and β3 in AG09319 cells were significantly higher than those in MC3T3-E1 cells. Conclusions: With the limitation of this study, we suggested that GRGD-modified chitosan, especially at high concentration, could enhance the growth of various periodontal

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

Science.gov (United States)

Cantrell, A R; Scheuer, T; Catterall, W A

1999-07-01

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

Synthesis of water soluble glycine capped silver nanoparticles and their surface selective interaction

International Nuclear Information System (INIS)

Agasti, Nityananda; Singh, Vinay K.; Kaushik, N.K.

2015-01-01

Highlights: • Synthesis of water soluble silver nanoparticles at ambient reaction conditions. • Glycine as stabilizing agent for silver nanoparticles. • Surface selective interaction of glycine with silver nanoparticles. • Glycine concentration influences crystalinity and optical property of silver nanoparticles. - Abstract: Synthesis of biocompatible metal nanoparticles has been an area of significant interest because of their wide range of applications. In the present study, we have successfully synthesized water soluble silver nanoparticles assisted by small amino acid glycine. The method is primarily based on reduction of AgNO 3 with NaBH 4 in aqueous solution under atmospheric air in the presence of glycine. UV–vis spectroscopy, transmission electron microscopy (TEM), X–ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG) and differential thermal analysis (DTA) techniques used for characterization of resulting silver nanoparticles demonstrated that, glycine is an effective capping agent to stabilize silver nanoparticles. Surface selective interaction of glycine on (1 1 1) face of silver nanoparticles has been investigated. The optical property and crystalline behavior of silver nanoparticles were found to be sensitive to concentration of glycine. X–ray diffraction studies ascertained the phase specific interaction of glycine on silver nanoparticles. Silver nanoparticles synthesized were of diameter 60 nm. We thus demonstrated an efficient synthetic method for synthesis of water soluble silver nanoparticles capped by amino acid under mild reaction conditions with excellent reproducibility

High-Dose Glycine Treatment of Refractory Obsessive-Compulsive Disorder and Body Dysmorphic Disorder in a 5-Year Period

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W. Louis Cleveland

2009-01-01

Full Text Available This paper describes an individual who was diagnosed with obsessive-compulsive disorder (OCD and body dysmorphic disorder (BDD at age 17 when education was discontinued. By age 19, he was housebound without social contacts except for parents. Adequate trials of three selective serotonin reuptake inhibitors, two with atypical neuroleptics, were ineffective. Major exacerbations following ear infections involving Group A -hemolytic streptococcus at ages 19 and 20 led to intravenous immune globulin therapy, which was also ineffective. At age 22, another severe exacerbation followed antibiotic treatment for H. pylori. This led to a hypothesis that postulates deficient signal transduction by the N-methyl-D-aspartate receptor (NMDAR. Treatment with glycine, an NMDAR coagonist, over 5 years led to robust reduction of OCD/BDD signs and symptoms except for partial relapses during treatment cessation. Education and social life were resumed and evidence suggests improved cognition. Our findings motivate further study of glycine treatment of OCD and BDD.

First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

DEFF Research Database (Denmark)

Hansen, Gert Helge; Belhage, B; Schousboe, A

1992-01-01

Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect imm...

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Science.gov (United States)

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Increased transient receptor potential vanilloid type 1 (TRPV1) channel expression in hypertrophic heart

DEFF Research Database (Denmark)

Thilo, Florian; Liu, Ying; Schulz, Nico

2010-01-01

The aim of this study was to compare the expression of transient receptor potential vanilloid type 1 (TRPV1) channels in hypertrophic hearts from transgenic mice showing overexpression of the catalytic subunit alpha of protein phosphatase 2A alpha (PP2Ac alpha) with wild-type mice and with TRPV1-...... alpha transgenic mice compared to wild-type mice and TRPV1-/- mice (8.6±1.3mg/g; 5.4±0.3mg/g; and 5.4±0.4mg/g; respectively; p...

Molecular characterisation of a recombinant bovine glycine N-acyltransferase / Christoffel Petrus Stephanus Badenhorst

OpenAIRE

Badenhorst, Christoffel Petrus Stephanus

2010-01-01

Conjugation of glycine to organic acids is an important detoxification mechanism. Metabolites of aspirin and industrial solvents, benzoic acid found in plant material and many endogenous metabolites are detoxified by conjugation to glycine. The enzyme responsible for glycine conjugation, glycine N-acyltransferase (GL YAT), is investigated in this study. The enzyme is also important for the management of organic acidemias which are inherited metabolic diseases. However, not all ...

Interaction of Heterodera glycines and Glomus mosseae on Soybean.

Science.gov (United States)

Todd, T C; Winkler, H E; Wilson, G W

2001-12-01

The effects of the arbuscular mycorrhizal (AM) fungus Glomus mosseae on Heterodera glycines-soybean interactions were investigated in greenhouse experiments. Mycorrhizal and nonmycorrhizal soybean cultivars that were either resistant or susceptible to H. glycines were exposed to initial nematode population densities (Pi) of 0, 100, 1,000, or 10,000 eggs and infective juveniles. Soybean growth, nematode reproduction, and AM fungal colonization were determined after 35 (experiment I) and 83 (experiment II) days. Soybean shoot and root weights were reduced an average 29% across H. glycines Pi but were 36% greater overall in the presence of G. mosseae. Analyses of variance indicated that root colonization and stimulation of soybean growth by G. mosseae were inhibited at high H. glycines Pi, while the combined effects of the nematode and fungus on soybean growth were best described as additive in linear regression models. No evidence for increased nematode tolerance of mycorrhizal soybean plants was observed. Nematode population densities and reproduction were lower on a nematode-resistant soybean cultivar than on a susceptible cultivar, but reproduction was comparable on mycorrhizal and nonmycorrhizal plants. Root colonization by G. mosseae was reduced at high nematode Pi. The results suggest that nematode antagonism to the mycorrhizal symbiosis is a more likely consequence of interactions between H. glycines and AM fungi on soybean than is nematode suppression by the fungus.

Nitrogen functionality of glucose-glycine condensate; Glucose to glycine tono shukugo hanno (shukugobutsuchu no chisso kagobutsu no keitai bunseki)

Energy Technology Data Exchange (ETDEWEB)

Tanaka, C.; Yoshioka, T.; Komano, T.; Mashimo, K.; Wainai, T. [Nihon University, Tokyo (Japan). College of Science and Technology; Sugimoto, Y.; : Miki, Y. [National Institute of Materials and Chemical Research, Tsukuba (Japan)

1996-10-28

In order to clarify a humification process in the early stage of coalification, the nitrogen functionality of prepared glucose-glycine condensate was studied experimentally. In experiment, the condensate was prepared by heating the mixture of glucose, glycine and water in a autoclave at 130{degree}C for 50 hours, and furthermore heating the produced solid material in water at 300{degree}C. After the condensate was hydrocracked, the fraction, condensate and hydrocracking residue were analyzed by elementary analyzer, {sup 13}C-NMR, XPS, FT-IR, capillary GC-FID/NPD and GC-MS. As a result, the glucose-glycine condensate could be arranged on the basis of three types of nitrogen such as pyridine, pyrrole and quaternary amine type. Pyridine type nitrogen increased, while quaternary amine type one decreased with an increase in heating treatment temperature. Rich pyrrole type nitrogen and poor pyridine type one were found in light nitrogen compounds in hydrocracked products. 2 refs., 4 figs., 2 tabs.

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

Science.gov (United States)

Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

Loss of Transient Receptor Potential Ankyrin 1 Channel Deregulates Emotion, Learning and Memory, Cognition, and Social Behavior in Mice.

Science.gov (United States)

Lee, Kuan-I; Lin, Hui-Ching; Lee, Hsueh-Te; Tsai, Feng-Chuan; Lee, Tzong-Shyuan

2017-07-01

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel that helps regulate inflammatory pain sensation and nociception and the development of inflammatory diseases. However, the potential role of the TRPA1 channel and the underlying mechanism in brain functions are not fully resolved. In this study, we demonstrated that genetic deletion of the TRPA1 channel in mice or pharmacological inhibition of its activity increased neurite outgrowth. In vivo study in mice provided evidence of the TRPA1 channel as a negative regulator in hippocampal functions; functional ablation of the TRPA1 channel in mice enhanced hippocampal functions, as evidenced by less anxiety-like behavior, and enhanced fear-related or spatial learning and memory, and novel location recognition as well as social interactions. However, the TRPA1 channel appears to be a prerequisite for motor function; functional loss of the TRPA1 channel in mice led to axonal bundle fragmentation, downregulation of myelin basic protein, and decreased mature oligodendrocyte population in the brain, for impaired motor function. The TRPA1 channel may play a crucial role in neuronal development and oligodendrocyte maturation and be a potential regulator in emotion, cognition, learning and memory, and social behavior.

Modulation of voltage-gated Ca2+ channels by G protein-coupled receptors in celiac-mesenteric ganglion neurons of septic rats.

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Mohamed Farrag

Full Text Available Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca2+ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca2+ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca2+ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE-mediated Ca2+ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca2+ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca2+ channel modulation.

Glycine facilitates gamma-glutamylcysteinylethyl ester-mediated increase in liver glutathione level.

Science.gov (United States)

Nishida, K; Ohta, Y; Ishiguro, I

1997-08-27

gamma-Glutamylcysteinylethyl ester (gamma-GCE) increases reduced glutathione (GSH) levels in GSH-depleted rat hepatocytes. Because glycine, a constituent of GSH, exists at 0.3 to 0.4 mM in rat plasma, we examined the influence of glycine added to the medium on the action of gamma-GCE to increase GSH levels in the rat hepatocytes. Glycine (0.2-0.8 mM) dose-dependently enhanced gamma-GCE-mediated increase in intracellular GSH levels with an increase in intracellular gamma-GCE levels. These results indicate that exogenous glycine facilitates gamma-GCE-mediated increase in intracellular GSH levels in rat hepatocytes possibly by enhancing the uptake of gamma-GCE into the cells.

Impaired absorption of marked oligopeptide Glycine-I Tyrosine-Glycine after successful autologous-allotopic ileal mucosa transplantation in beagles.

Science.gov (United States)

Beiler, H A; Steinorth, J; Witt, A; Mier, W; Mohammed, A; Waag, K L; Zachariou, Z

2004-10-01

After establishing a method for ileal mucosa transplantation in an animal model, the authors investigated the absorptive capacity for oligopeptides of the transplanted mucosa. In 14 beagle dogs the authors transplanted ileal mucosa in a vascularized demucosed segment of the transverse colon. The colonic wall-ileal mucosa complex then was integrated in the ileal continuity. Six animals were lost owing to operative complications. Absorptive capacity for oligopeptides was measured in the remaining 8 animals with the iodine 131 (131I)-marked tripeptide glycine-tyrosine-glycine before and 4 weeks after transplantation. The results were compared and analyzed with the Student's t test for matched pairs. Blood concentrations of the marked tripeptide with P value less than .05 were considered as a significant reduction in the absorptive capacity of the transplanted ileal mucosa. After fixation with glutaraldehyd graft, uptake of the colonic wall-ileal mucosa complex was evaluated histologically in 8 animals. In all 8 animals, a 100% graft uptake was verified in all sections. Fifteen minutes after application of 15 MBc Glycine-131I-Tyrosine-Glycine there was no significant difference in the absorption between normal and transplanted ileal mucosa. After 30 minutes, the absorption of the transplanted ileal mucosa showed a tendency (P < .1) for an impaired uptake of the marked tripeptide. However, 60 minutes after application the difference in the absorptive capacity of the transplanted ileal mucosa was significant (P < .05). Autologous allotopic ileal mucosa transplantation is feasible; however, an impaired absorption of oligopeptides of the transplanted mucosa 4 weeks after transplantation could be observed.

Post-Translational Modifications of TRP Channels

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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.

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

OpenAIRE

Shang, Lijun; Tucker, Stephen J.

2007-01-01

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K+ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the ?helix-bundle crossing?. However, in the inwardly rectifying (Kir) potassium channel family, the role of this ?hinge? residue in the second transmembrane domain (TM2) and t...

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

Science.gov (United States)

Mostyn, Shannon N; Carland, Jane E; Shimmon, Susan; Ryan, Renae M; Rawling, Tristan; Vandenberg, Robert J

2017-09-20

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC 50 of 9 μM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 μM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Light intensity affects the uptake and metabolism of glycine by pakchoi (Brassica chinensis L.)

Science.gov (United States)

Ma, Qingxu; Cao, Xiaochuang; Wu, Lianghuan; Mi, Wenhai; Feng, Ying

2016-02-01

The uptake of glycine by pakchoi (Brassica chinensis L.), when supplied as single N-source or in a mixture of glycine and inorganic N, was studied at different light intensities under sterile conditions. At the optimal intensity (414 μmol m-2 s-1) for plant growth, glycine, nitrate, and ammonium contributed 29.4%, 39.5%, and 31.1% shoot N, respectively, and light intensity altered the preferential absorption of N sources. The lower 15N-nitrate in root but higher in shoot and the higher 15N-glycine in root but lower in shoot suggested that most 15N-nitrate uptake by root transported to shoot rapidly, with the shoot being important for nitrate assimilation, and the N contribution of glycine was limited by post-uptake metabolism. The amount of glycine that was taken up by the plant was likely limited by root uptake at low light intensities and by the metabolism of ammonium produced by glycine at high light intensities. These results indicate that pakchoi has the ability to uptake a large quantity of glycine, but that uptake is strongly regulated by light intensity, with metabolism in the root inhibiting its N contribution.

A kinetic model for the glucose/glycine Maillard reaction pathways

NARCIS (Netherlands)

Martins, S.I.F.S.; Boekel, van M.A.J.S.

2005-01-01

A comprehensive kinetic model for the glucose/glycine Maillard reaction is proposed based on an approach called multiresponse kinetic modelling. Special attention was paid to reactants, intermediates and end products: -fructose, N-(1-deoxy--fructos-1-yl)-glycine (DFG), 1-deoxy-2,3-hexodiulose and

NMDA receptor channels: labeling of MK-801 with iodine-125 and fluorine-18

International Nuclear Information System (INIS)

Wieland, D.M.; Kilbourn, M.R.; Yang, D.J.; Laborde, E.; Gildersleeve, D.L.; Van Dort, M.E.; Pirat, J.-L.; Ciliax, B.J.; Young, A.B.

1988-01-01

Methods for labeling the glutamate channel blocking agent MK-801 with iodine-125 ( 125 I) and fluorine-18 ( 18 F) are described. Radioiodine was incorporated in the 1- or 3-positions of the aromatic ring of (±)MK-801 by solid-state halogen exchange techniques. Attachment of the [ 18 F]fluoromethyl group to the bridgehead methyl position was achieved by reaction of [ 18 F]fluoride with the triflamide alcohol or the novel cyclic sulfamate recently reported by Merck chemists. Radiochemical yields of (±)13-[ 18 F]-fluoromethyl-MK-801 were >72%, EOB; radiochemical purity > 99%. In competitive binding studies using rat brain homogenates, (±)3-bromo-MK-801 showed greater affinity than (±)MK-801 for the glutamate-linked channel. The experimental log P (2.1 ± 0.1) of MK-801 is optimal for transit of the blood-brain barrier. These preliminary findings support further testing of [ 123 I]iodo-MK-801 and [ 18 F]fluoromethyl-MK-801 as possible agents for in vivo mapping of the glutamate receptor complex. (author)

P2X receptor-ion channels in the inflammatory response in adipose tissue and pancreas-potential triggers in onset of type 2 diabetes?

DEFF Research Database (Denmark)

Novak, Ivana; Solini, Anna

2018-01-01

-cell and adipose tissue. In the former, P2Y and possibly some P2X receptors-ion channels regulate insulin secretion, but it is still debated whether excessive ATP can via P2X receptors impair β-cell function directly or whether cell damage is due to an excessive systemic release of cytokines. In human adipocytes......, the P2X7 receptor promotes the release of inflammatory cytokines, at least in part via inflammasome activation, likely contributing to systemic insulin resistance. This receptor-inflammasome system is also strongly activated in macrophages infiltrating both pancreas and adipose tissue, mediating...

Rapid Methods to Distinguish Heterodera schachtii from Heterodera glycines Using PCR Technique

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Hyoung Rai Ko

2017-09-01

Full Text Available The purpose of this study was to develop rapid methods for distinguishing between Heterodera schachtii and H. glycines detected from chinese cabbage fields of highland in Gangwon, Korea. To do this, we performed PCR-RFLP and PCR with the primers set developed in this study for GC147, GC408 and PM001 population, H. schachtii, and YS224, DA142 and BC115 population, H. glycines. Eight restriction enzymes generated RFLP profiles of mtDNA COI region for populations of H. schachtii and H. glycines, repectively. As a result, treatment of two restriction enzymes, RsaI and HinfI, were allowed to distinguish H. schachtii from H. glycines based on the differences of DNA band patterns. The primer set, #JBS1, #JBG1 and #JB3R, amplified specific fragments with 277 and 339 bp of H. schachtii, 339 bp of H. glycines, respectively, while it did not amplify fragments from three root-knot nematodes and two root-lesion nematodes. Thus, the primer set developed in this study could be a good method, which is used to distinguish between H. schachtii and H. glycines.

Slack, Slick, and Sodium-Activated Potassium Channels

Science.gov (United States)

Kaczmarek, Leonard K.

2013-01-01

The Slack and Slick genes encode potassium channels that are very widely expressed in the central nervous system. These channels are activated by elevations in intracellular sodium, such as those that occur during trains of one or more action potentials, or following activation of nonselective cationic neurotransmitter receptors such as AMPA receptors. This review covers the cellular and molecular properties of Slack and Slick channels and compares them with findings on the properties of sodium-activated potassium currents (termed KNa currents) in native neurons. Human mutations in Slack channels produce extremely severe defects in learning and development, suggesting that KNa channels play a central role in neuronal plasticity and intellectual function. PMID:24319675

A DFT study of adsorption of glycine onto the surface of BC_2N nanotube

International Nuclear Information System (INIS)

Soltani, Alireza; Azmoodeh, Zivar; Javan, Masoud Bezi; Lemeski, E. Tazikeh; Karami, Leila

2016-01-01

Highlights: • Glycine adsorption over the pristine BC_2N nanotubes is investigated by DFT calculations. • Adsorption of glycine in its zwitterionic form is stronger in comparison with the radical form. • Adsorption of glycine from its amine head on adsorbent leads to a significant decrease in the electronic properties. - Abstract: A theoretical study of structure and the energy interaction of amino acid glycine (NH_2CH_2COOH) with BC_2N nanotube is crucial for apperception behavior occurring at the nanobiointerface. Herein, we studied the adsorption of glycine in their radical and zwitterionic forms upon the surface of BC_2N nanotube using M06 functional and 6-311G** standard basis set. We also considered the different orientations of the glycine amino acid on the surface of adsorbent. Further, we found out that the stability of glycine from its carbonyl group is higher than hydroxyl and amine groups. Our results also indicated that the electronic structure of BC_2N nanotube on the adsorption of glycine from its amine group is more altered than the other groups. Our study exhibits that opto-electronic property of adsorbent is changed after the glycine adsorption.

Zwitterionization of glycine in water environment: Stabilization mechanism and NMR spectral signatures

Science.gov (United States)

Valverde, Danillo; da Costa Ludwig, Zélia Maria; da Costa, Célia Regina; Ludwig, Valdemir; Georg, Herbert C.

2018-01-01

At physiological conditions, myriads of biomolecules (e.g., amino acids, peptides, and proteins) exist predominantly in the zwitterionic structural form and their biological functions will result in these conditions. However these geometrical structures are inaccessible energetically in the gas phase, and at this point, stabilization of amino-acids in physiological conditions is still under debate. In this paper, the electronic properties of a glycine molecule in the liquid environment were studied by performing a relaxation of the glycine geometry in liquid water using the free energy gradient method combined with a sequential quantum mechanics/molecular mechanics approach. A series of Monte Carlo Metropolis simulations of the glycine molecule embedded in liquid water, followed by only a quantum mechanical calculation in each of them were carried out. Both the local and global liquid environments were emphasized to obtain nuclear magnetic resonance (NMR) parameters for the glycine molecule in liquid water. The results of the equilibrium structure in solution and the systematic study of the hydrogen bonds were used to discard the direct proton transfer from the carboxyl group to the ammonium group of the glycine molecule in water solution. The calculations of the Density Functional Theory (DFT) were performed to study the polarization of the solvent in the parameters of nuclear magnetic resonance of the glycine molecule in liquid water. DFT calculations predicted isotropic chemical changes on the H, C, N, and O atoms of glycine in liquid water solution which agree with the available experimental data.

Crystallization of glycine with ultrasound

DEFF Research Database (Denmark)

Louhi-Kultanen, Marjatta; Karjalainen, Milja; Rantanen, Jukka

2006-01-01

Sonocrystallization has proved to be an efficient tool to influence the external appearance and structure of a crystalline product obtained by various crystallization methods. The present work focuses on high intensity sonocrystallization of glycine by varying amplitude of ultrasound with an ultr...... ultrasound power. This study also showed, the higher the ultrasound amplitude the smaller the crystals obtained.......Sonocrystallization has proved to be an efficient tool to influence the external appearance and structure of a crystalline product obtained by various crystallization methods. The present work focuses on high intensity sonocrystallization of glycine by varying amplitude of ultrasound...... with an ultrasound frequency of 20kHz at two temperature ranges 40-50 and 20-30 degrees C in a jacketed 250-ml cooling crystallizer equipped with a stirrer. The polymorph composition of the obtained crystals was analyzed with a temperature variable X-ray powder diffractometer (XRPD). XRPD results showed that...

Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel.

Science.gov (United States)

Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

2012-01-06

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

Kinetic study of carbon dioxide absorption into glycine promoted diethanolamine (DEA)

Science.gov (United States)

Pudjiastuti, Lily; Susianto, Altway, Ali; IC, Maria Hestia; Arsi, Kartika

2015-12-01

In industry, especially petrochemical, oil and natural gas industry, required separation process of CO2 gas which is a corrosive gas (acid gas). This characteristic can damage the plant utility and piping systems as well as reducing the caloric value of natural gas. Corrosive characteristic of CO2 will appear in areas where there is a decrease in temperature and pressure, such as at the elbow pipe, tubing, cooler and injector turbine. From disadvantages as described above, then it is important to do separation process in the CO2 gas stream, one of the method for remove CO2 from the gas stream is reactive absorption using alkanolamine based solution with promotor. Therefore, this study is done to determine the kinetics constant of CO2 absorption in diethanolamine (DEA) solution using a glycine promoter. Glycine is chosen as a promoter because glycine is a primary amine compound which is reactive, moreover, glycine has resistance to high temperatures so it will not easy to degradable and suitable for application in industry. The method used in this study is absorption using laboratory scale wetted wall column equipment at atmospheric of pressure. This study will to provide the reaction kinetics data information in order to optimize the separation process of CO2 in the industrialized world. The experimental results show that rising temperatures from 303,15 - 328,15 K and the increase of concentration of glycine from 1% - 3% weight will increase the absorption rate of carbon dioxide in DEA promoted with glycine by 24,2% and 59,764% respectively, also the reaction kinetic constant is 1.419 × 1012 exp (-3634/T) (m3/kmol.s). This result show that the addition of glycine as a promoter can increase absorption rate of carbon dioxide in diethanolamine solution and cover the weaknesses of diethanolamine solution.

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

Science.gov (United States)

Sato, Masaki; Ogura, Kazuhiro; Kimura, Maki; Nishi, Koichi; Ando, Masayuki; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2018-04-27

Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 -negative medium-sized neurons. Action potentials in these isolectin B 4 -negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels

Czech Academy of Sciences Publication Activity Database

Bertaso, F.; Lill, Y.; Airas, J.M.; Espeut, J.; Blahoš, Jaroslav; Bockaert, J.; Fagni, L.; Betz, H.; Far, O.E.

2006-01-01

Roč. 99, - (2006), s. 288-298 ISSN 0022-3042 R&D Projects: GA ČR GA204/05/0920 Institutional research plan: CEZ:AV0Z50390512 Keywords : calmodulin * metabotropic glutamate receptor * calcium channel Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.260, year: 2006

L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli.

Science.gov (United States)

Song, Hun-Suk; Jeon, Jong-Min; Choi, Yong Keun; Kim, Jun-Young; Kim, Wooseong; Yoon, Jeong-Jun; Park, Kyungmoon; Ahn, Jungoh; Lee, Hongweon; Yang, Yung-Hun

2017-12-28

Lignocellulose is now a promising raw material for biofuel production. However, the lignin complex and crystalline cellulose require pretreatment steps for breakdown of the crystalline structure of cellulose for the generation of fermentable sugars. Moreover, several fermentation inhibitors are generated with sugar compounds, majorly furfural. The mitigation of these inhibitors is required for the further fermentation steps to proceed. Amino acids were investigated on furfural-induced growth inhibition in E. coli producing isobutanol. Glycine and serine were the most effective compounds against furfural. In minimal media, glycine conferred tolerance against furfural. From the IC₅₀ value for inhibitors in the production media, only glycine could alleviate growth arrest for furfural, where 6 mM glycine addition led to a slight increase in growth rate and isobutanol production from 2.6 to 2.8 g/l under furfural stress. Overexpression of glycine pathway genes did not lead to alleviation. However, addition of glycine to engineered strains blocked the growth arrest and increased the isobutanol production about 2.3-fold.

Cerebrovascular endothelin-1 hyper-reactivity is associated with transient receptor potential canonical channels 1 and 6 activation and delayed cerebral hypoperfusion after forebrain ischaemia in rats

DEFF Research Database (Denmark)

Johansson, S E; Andersen, X E D R; Hansen, R H

2015-01-01

. METHODS: Experimental forebrain ischaemia was induced in Wistar male rats by a two-vessel occlusion model, and the cerebral blood flow was measured by magnetic resonance imaging two days after reperfusion. In vitro vasoreactivity studies, immunofluorescence and quantitative PCR were performed on cerebral...... in the vascular smooth muscle cells was enhanced and correlated with decreased cerebral blood flow two days after forebrain ischaemia. Furthermore, under conditions when voltage-dependent calcium channels were inhibited, endothelin-1-induced cerebrovascular contraction was enhanced and this enhancement...... was presumably mediated by Ca(2+) influx via upregulated transient receptor potential canonical channels 1 and 6. CONCLUSIONS: Our data demonstrates that endothelin-1-mediated influx of extracellular Ca(2+) activates transient receptor potential canonical channels 1 and 6 in cerebral vascular smooth muscle cells...

Sublethal and hormesis effects of imidacloprid on the soybean aphid Aphis glycines.

Science.gov (United States)

Qu, Yanyan; Xiao, Da; Li, Jinyu; Chen, Zhou; Biondi, Antonio; Desneux, Nicolas; Gao, Xiwu; Song, Dunlun

2015-04-01

The soybean aphid, Aphis glycines Matsumura, is a major pest in soybean crop. Current management of this pest relies mainly on insecticides applications, and the neonicotinoid imidacloprid has been proposed as an effective insecticide to control A. glycines in soybean field. Imidacloprid at lethal concentrations not only exerts acute toxicity to A. glycines, but also cause various biological changes when aphids are chronically exposed to lower concentrations. In this study, we assessed the effects of a low-lethal (0.20 mg L(-1)) and two sublethal (0.05 and 0.10 mg L(-1)) imidacloprid concentrations on various A. glycines life history traits. Aphid exposure to 0.20 mg L(-1) imidacloprid caused slower juvenile development, shorter reproductive period, and reduced adult longevity, fecundity and total lifespan. Stimulatory effects, i.e. hormesis, on reproduction and immature development duration were observed in aphids exposed to the lower sublethal imidacloprid concentrations. Consequently, the net reproduction rate (R 0) was significantly higher than in the control aphids. These findings stress the importance of the actual imidacloprid concentration in its toxicological properties on A. glycines. Therefore, our results would be useful for assessing the overall effects of imidacloprid on A. glycines and for optimizing integrated pest management programs targeting this pest.

Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides

Science.gov (United States)

Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

2015-01-01

Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor PI(4)P from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 or PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. PMID:25670203

The effects of glycine on subjective daytime performance in partially sleep-restricted healthy volunteers

Directory of Open Access Journals (Sweden)

Makoto eBannai

2012-04-01

Full Text Available Approximately 30% of the general population suffers from insomnia. Given that insomnia causes many problems, amelioration of the symptoms is crucial. Recently, we found that a nonessential amino acid, glycine subjectively and objectively improves sleep quality in humans who have difficulty sleeping. We evaluated the effects of glycine on daytime sleepiness, fatigue and performances in sleep-restricted healthy subjects. Sleep was restricted to 25% less than the usual sleep time for three consecutive nights. Before bedtime, 3 g of glycine or placebo were ingested, sleepiness and fatigue were evaluated using the visual analogue scale (VAS and a questionnaire, and performance were estimated by personal computer (PC performance test program on the following day. In subjects given glycine, the VAS data showed a significant reduction in fatigue and a tendency toward reduced sleepiness. These observations were also found via the questionnaire, indicating that glycine improves daytime sleepiness and fatigue induced by acute sleep restriction. PC performance test revealed significant improvement in psychomotor vigilance test. We also measured plasma melatonin and the expression of circadian-modulated genes expression in the rat suprachiasmatic nucleus (SCN to evaluate the effects of glycine on circadian rhythms. Glycine did not show significant effects on plasma melatonin concentrations during either the dark or light period. Moreover, the expression levels of clock genes such as Bmal1 and Per2 remained unchanged. However, we observed a glycine-induced increase in the neuropeptides arginine vasopressin and vasoactive intestinal polypeptide in the light period. Although no alterations in the circadian clock itself were observed, our results indicate that glycine modulated SCN function. Thus, glycine modulates certain neuropeptides in the SCN and this phenomenon may indirectly contribute to improving the occasional sleepiness and fatigue induced by sleep

In vivo binding and autoradiographic imaging of (+)-3-[125I]Iodo-MK-801 to the NMDA receptor-channel complex in rat brain

International Nuclear Information System (INIS)

Gibson, R.E.; Burns, H.D.; Thorpe, H.H.; Waisi Eng; Ransom, R.; Solomon, H.

1992-01-01

Radioiodinated (+)-3-Iodo-MK-801 is a high affinity radioligand for the N-methyl-D-aspartate (NMDA) receptor-channel complex. We have demonstrated in vivo localization in the CNS of rat which is stereoselective and blocked by coinjection of unlabeled MK-801. Autoradiography indicates localization in vivo which is in concordance with in vitro autoradiographic studies. These results indicate that radioiodinated (+)-3-Iodo-MK-801 is a useful probe for in vitro and in vivo autoradiographic studies and suggest that radioligands for the NMDA receptor may be developed which will provide in vivo images of receptor distribution in man. (author)

Localization of high affinity [3H]glycine transport sites in the cerebellar cortex

International Nuclear Information System (INIS)

Wilkin, G.P.; Csillag, A.; Balazs, R.; Kingsbury, A.E.; Wilson, J.E.; Johnson, A.L.

1981-01-01

A study was made of [ 3 H ]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [ 3 H]glycine at the low concentration used. Glial cells also took up [ 3 H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [ 3 H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [ 3 H]GABA uptake (Ksub(i) over 600 μM vs the Ksub(t) of about 20 μM) and that GABA is an even weaker inhibitor of [ 3 H]glycine uptake. (Auth.)

Effect of Glycine on Lead Mobilization, Lead-Induced Oxidative Stress, and Hepatic Toxicity in Rats

Directory of Open Access Journals (Sweden)

Yolanda Alcaraz-Contreras

2011-01-01

Full Text Available The effectiveness of glycine in treating experimental lead intoxication was examined in rats. Male Wistar rats were exposed to 3 g/L lead acetate in drinking water for 5 weeks and treated thereafter with glycine (100 and 500 mg/kg, orally once daily for 5 days or glycine (1000 mg/kg, orally once daily for 28 days. The effect of these treatments on parameters indicative of oxidative stress (glutathione and malondialdehyde levels, the activity of blood -aminolevulinic acid dehydratase, and lead concentration in blood, liver, kidney, brain, and bone were investigated. Liver samples were observed for histopathological changes. Glycine was found to be effective in (1 increasing glutathione levels; (2 reducing malondialdehyde levels; (3 decreasing lead levels in bone with the highest dose. However, glycine had no effect on lead mobilization when 100 and 500 mg/kg glycine were administered. In microscopic examination, glycine showed a protective effect against lead intoxication.

Alteration of glycine receptor immunoreactivity in the auditory brainstem of mice following three months of exposure to radiofrequency radiation at SAR 4.0 W/kg.

Science.gov (United States)

Maskey, Dhiraj; Kim, Hyung Gun; Suh, Myung-Whan; Roh, Gu Seob; Kim, Myeung Ju

2014-08-01

The increasing use of mobile communication has triggered an interest in its possible effects on the regulation of neurotransmitter signals. Due to the close proximity of mobile phones to hearing-related brain regions during usage, its use may lead to a decrease in the ability to segregate sounds, leading to serious auditory dysfunction caused by the prolonged exposure to radiofrequency (RF) radiation. The interplay among auditory processing, excitation and inhibitory molecule interactions plays a major role in auditory function. In particular, inhibitory molecules, such a glycine, are predominantly localized in the auditory brainstem. However, the effects of exposure to RF radiation on auditory function have not been reported to date. Thus, the aim of the present study was to investigate the effects of exposure to RF radiation on glycine receptor (GlyR) immunoreactivity (IR) in the auditory brainstem region at 835 MHz with a specific absorption rate of 4.0 W/kg for three months using free-floating immunohistochemistry. Compared with the sham control (SC) group, a significant loss of staining intensity of neuropils and cells in the different subdivisions of the auditory brainstem regions was observed in the mice exposed to RF radiation (E4 group). A decrease in the number of GlyR immunoreactive cells was also noted in the cochlear nuclear complex [anteroventral cochlear nucleus (AVCN), 31.09%; dorsal cochlear nucleus (DCN), 14.08%; posteroventral cochlear nucleus (PVCN), 32.79%] and the superior olivary complex (SOC) [lateral superior olivary nucleus (LSO), 36.85%; superior paraolivary nucleus (SPN), 24.33%, medial superior olivary nucleus (MSO), 23.23%; medial nucleus of the trapezoid body (MNTB), 10.15%] of the mice in the E4 group. Auditory brainstem response (ABR) analysis also revealed a significant threshold elevation of in the exposed (E4) group, which may be associated with auditory dysfunction. The present study suggests that the auditory brainstem region

Synthesis of Diopside by Solution Combustion Process Using Glycine Fuel

Science.gov (United States)

Sherikar, Baburao N.; Umarji, A. M.

Nano ceramic Diopside (CaMgSi2O6) powders are synthesized by Solution Combustion Process(SCS) using Calcium nitrate, Magnesium nitrate as oxidizer and glycine as fuel, fumed silica as silica source. Ammonium nitrate (AN) is used as extra oxidizer. Effect of AN on Diopside phase formation is investigated. The adiabatic flame temperatures are calculated theoretically for varying amount of AN according to thermodynamic concept and correlated with the observed flame temperatures. A “Multi channel thermocouple setup connected to computer interfaced Keithley multi voltmeter 2700” is used to monitor the thermal events during the process. An interpretation based on maximum combustion temperature and the amount of gases produced during reaction for various AN compositions has been proposed for the nature of combustion and its correlation with the characteristics of as synthesized powder. These powders are characterized by XRD, SEM showing that the powders are composed of polycrystalline oxides with crystallite size of 58nm to 74nm.

Kinetics and mechanism of oxidation of glycine by iron(III)

Indian Academy of Sciences (India)

Kinetics and mechanism of oxidation of glycine by iron(III)-1,10-phenanthroline complex has been studied in perchloric acid medium. The reaction is first order with respect to iron(III) and glycine. An increase in (phenanthroline) increases the rate, while increase in [H+] decreases the rate. Hence it can be inferred that the ...

Role of Transient Receptor Potential Ankyrin 1 Ion Channel and Somatostatin sst4 Receptor in the Antinociceptive and Anti-inflammatory Effects of Sodium Polysulfide and Dimethyl Trisulfide

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István Z. Bátai

2018-02-01

Full Text Available Transient receptor potential ankyrin 1 (TRPA1 non-selective ligand-gated cation channels are mostly expressed in primary sensory neurons. Polysulfides (POLYs are Janus-faced substances interacting with numerous target proteins and associated with both protective and detrimental processes. Activation of TRPA1 in sensory neurons, consequent somatostatin (SOM liberation and action on sst4 receptors have recently emerged as mediators of the antinociceptive effect of organic trisulfide dimethyl trisulfide (DMTS. In the frame of the present study, we set out to compare the participation of this mechanism in antinociceptive and anti-inflammatory effects of inorganic sodium POLY and DMTS in carrageenan-evoked hind-paw inflammation. Inflammation of murine hind paws was induced by intraplantar injection of carrageenan (3% in 30 µL saline. Animals were treated intraperitoneally with POLY (17 µmol/kg or DMTS (250 µmol/kg or their respective vehicles 30 min prior paw challenge and six times afterward every 60 min. Mechanical pain threshold and swelling of the paws were measured by dynamic plantar aesthesiometry and plethysmometry at 2, 4, and 6 h after initiation of inflammation. Myeloperoxidase (MPO activity in the hind paws were detected 6 h after challenge by luminescent imaging. Mice genetically lacking TRPA1 ion channels, sst4 receptors and their wild-type counterparts were used to examine the participation of these proteins in POLY and DMTS effects. POLY counteracted carrageenan-evoked mechanical hyperalgesia in a TRPA1 and sst4 receptor-dependent manner. POLY did not influence paw swelling and MPO activity. DMTS ameliorated all examined inflammatory parameters. Mitigation of mechanical hyperalgesia and paw swelling by DMTS were mediated through sst4 receptors. These effects were present in TRPA1 knockout animals, too. DMTS inhibited MPO activity with no participation of the sensory neuron–SOM axis. While antinociceptive effects of

Equilibria in aqueous cadmium-chloroacetate-glycinate systems. A convolution-deconvolution cyclic voltammetric study

International Nuclear Information System (INIS)

Abdel-Hamid, R.; Rabia, M.K.M.

1994-01-01

Stability constants and composition of cadmium-glycinate binary complexes were determined using cyclic voltammetry. Furthermore, binary and ternary complex equilibria for chloroacetates and glycinate with cadmium in 0.1 M aqueous KNO 3 at pH 10.4 and 298 K were investigated. Cadmium forms binary complexes with chloroacetates of low stability and ternary ones with chloroacetate-glycinate of significant stability. (author)

Secondary Emission From Synthetic Opal Infiltrated by Colloidal Gold and Glycine

International Nuclear Information System (INIS)

Dovbeshko, G.I.; Fesenko, O.M.; Boyko, V.V.; Romanyuk, V.R.; Gorelik, V.S.; Moiseyenko, V.N.; Sobolev, V.B.; Shvalagin, V.V.

2012-01-01

A comparison of the secondary emission (photoluminescence) and Bragg reflection spectra of photonic crystals (PC), namely, synthetic opals, opals infiltrated by colloidal gold, glycine, and a complex of colloidal gold with glycine is performed. The infiltration of colloidal gold and a complex of colloidal gold with glycine into the pores of PC causes a short-wavelength shift (about 5-15 nm) of the Bragg reflection and increases the intensity of this band by 1.5-3 times. In photoluminescence, the infiltration of PC by colloidal gold and colloidal gold with glycine suppresses the PC emission band near 375-450 nm and enhances the shoulder of the stop-zone band of PC in the region of 470-510 nm. The shape of the observed PC emission band connected with defects in synthetic opal is determined by the type of infiltrates and the excitation wavelength. Possible mechanisms of the effects are discussed.

Neuroactive Steroids: Receptor Interactions and Responses

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Kald Beshir Tuem

2017-08-01

Full Text Available Neuroactive steroids (NASs are naturally occurring steroids, which are synthesized centrally as de novo from cholesterol and are classified as pregnane, androstane, and sulfated neurosteroids (NSs. NASs modulate many processes via interacting with gamma-aminobutyric acid (GABA, N-methyl-d-aspartate, serotonin, voltage-gated calcium channels, voltage-dependent anion channels, α-adrenoreceptors, X-receptors of the liver, transient receptor potential channels, microtubule-associated protein 2, neurotrophin nerve growth factor, and σ1 receptors. Among these, NSs (especially allopregnanolone have high potency and extensive GABA-A receptors and hence demonstrate anticonvulsant, anesthetic, central cytoprotectant, and baroreflex inhibitory effects. NSs are also involved in mood and learning via serotonin and anti-nociceptive activity via T-type voltage-gated Ca2+ channels. Moreover, they are modulators of mitochondrial function, synaptic plasticity, or regulators of apoptosis, which have a role in neuroprotective via voltage-dependent anion channels receptors. For proper functioning, NASs need to be in their normal level, whereas excess and deficiency may lead to abnormalities. When they are below the normal, NSs could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit disorder could occur during excessive level. Overall, NASs are very important molecules with major neuropsychiatric activity.

Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

Science.gov (United States)

Mendum, Mary Lou; Smith, Linda Tombras

2002-01-01

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a Km of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a Km for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a Km for carnitine uptake of 1 to 3 μM and a Vmax of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected. PMID:12406761

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

DEFF Research Database (Denmark)

Nassini, Romina; Pedretti, Pamela; Moretto, Nadia

2012-01-01

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic...... inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express...... functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells...

Crystal lattice dependency of the free radicals found in irradiated glycine

NARCIS (Netherlands)

Bie, M.J.A. de; Braams, R.

1969-01-01

The EPR spectra, and hence the stable free radicals, are different for the - or γ-irradiated α-, β- and γ-crystal forms of polycrystalline glycone. Therefore comparisons of the trideutero-glycine EPR spectrum with the EPR spectra of non-deuterated glycine are open to question

Distribution profiles of transient receptor potential melastatin- and vanilloid-related channels in rat spermatogenic cells and sperm.

Science.gov (United States)

Li, Shilin; Wang, Xinghuan; Ye, Haixia; Gao, Weicheng; Pu, Xiaoyong; Yang, Zhonghua

2010-03-01

In the present study, we aimed to investigate the expression and distribution of transient receptor potential melastatin (TRPM)- and vanilloid (TRPV)- related channels in rat spermatogenic cells and spermatozoa. Spermatogenic cells and spermatozoa were obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of all TRPM and TRPV channel members with specific primers. Western blot analysis was applied for detecting the expression of TRPM and TRPV channel proteins. Immunohistochemistry staining for TRPM4, TRPM7 and TRPV5 was also performed in rat testis. The mRNAs of TRPM3, TRPM4, TRPM7 and TRPV5 were detected in the spermatogenic cells and spermatozoa in rat. Western blot analysis verified the expression of TRPM4, TRPM7 and TRPV5 in the rat spermatogenic cells and spermatozoa. Immunocytochemistry staining for TRPM and TRPV channel families indicated that TRPM4 and TRPM7 proteins were highly expressed in different stages of spermatogenic cells and spermatozoa, while TRPV5 protein was lowly expressed in these cells. Our results demonstrate that mRNAs or proteins for TRPM3, TRPM4, TRPM7 and TRPV5 exist in rat spermatogenic cells and spermatozoa. These data presented here may assist in elucidating the possible physiological function of TRPM and TRPV channels in spermatogenic cells and spermatozoa.

Ionotropic glutamate receptors (iGluRs) of the delta family (GluD1 ...

African Journals Online (AJOL)

Muhammad Zahid Khan

2016-10-20

Oct 20, 2016 ... GluD1 knockout mice (GluD1 KO) have normal learning in the Morris water maze .... could bind and activate the receptor.5,6 D-Ser and glycine have now been identified as .... English editing of this manuscript. References. 1.

Characterization and regulation of glycine transport in Fusarium oxysporum var. lini.

Science.gov (United States)

Castro, I M; Lima, A A; Nascimento, A F; Ruas, M M; Nicoli, J R; Brandão, R L

1996-08-01

Glycine was transported in Fusarium oxysporum cells, grown on glycine as the sole source of carbon and nitrogen, by a facilitated diffusion transport system with a half-saturation constant (Ks) of 11 mM and a maximum velocity (Vmax) of 1.2 mM (g dry weight)-1 h-1 at pH 5.0 and 26 degrees C. Under conditions of nitrogen starvation, the same system was present together with a high-affinity one (Ks) of about 47 microM and Vmax of about 60 microM (g dry weight)-1 h-1). The low-affinity system was more specific than the high-affinity system. Cells grown on gelatine showed the same behavior. In cells grown on glucose-gelatine medium, the low-affinity system was poorly expressed even after carbon and nitrogen starvation. Moreover, addition of glucose to cells grown on glycine and resuspended in mineral medium caused an increase of the glycine transport probably due to a boost in protein synthesis. This stimulation did not affect the Ks of the low-affinity system. These results demonstrate that, as is the case for other eukaryotic systems, F. oxysporum glycine transport is under control of nitrogen sources but its regulation by carbon sources appears to be more complex.

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...

Marine snail venoms: use and trends in receptor and channel neuropharmacology.

Science.gov (United States)

Favreau, Philippe; Stöcklin, Reto

2009-10-01

Venoms are rich mixtures of mainly peptides and proteins evolved by nature to catch and digest preys or for protection against predators. They represent extensive sources of potent and selective bioactive compounds that can lead to original active ingredients, for use as drugs, as pharmacological tools in research and for the biotechnology industry. Among the most fascinating venomous animals, marine snails offer a unique set of pharmacologically active components, targeting a wide diversity of receptors and ion channels. Recent advances still continue to demonstrate their huge neuropharmacological potential. In the quest for interesting pharmacological profiles, researchers face a vast number of venom components to investigate within time and technological constraints. A brief perspective on marine snail venom's complexity and features is given followed by the different discovery strategies and pharmacological approaches, exemplified with some recent developments. These advances will hopefully help further uncovering new pharmacologically important venom molecules.

Nereistoxin and cartap neurotoxicity attributable to direct block of the insect nicotinic receptor/channel.

Science.gov (United States)

Lee, Seog-Jong; Tomizawa, Motohiro; Casida, John E

2003-04-23

Nereistoxin (NTX) (4-dimethylamino-1,2-dithiolane) is the naturally occurring prototype for cartap [the bis(thiocarbamate) derivative of the NTX dithiol], which is generally regarded as a proinsecticide reverting to NTX. The aim of this study is to define the target site(s) for dithiolanes and dithiol esters. The affinity of [(3)H]NTX was not suitable for binding assays with honeybee (Apis mellifera) head membranes. However, NTX and cartap are equally potent, direct-acting, and competitive displacers of [(3)H]thienylcyclohexylpiperidine binding at the noncompetitive blocker (NCB) site of the Apis nicotinic acetylcholine receptor (nAChR)/channel. NTX also binds at the Apis [(3)H]imidacloprid agonist site, but cartap does not. As candidate metabolic pathways, sequential N-desmethylation and S-oxidation of NTX progressively reduce its potency at the NCB site and toxicity to houseflies. A P450 inhibitor reduces the toxicity of NTX and enhances it with cartap. Surprisingly, cartap is not just a pro-NTX but instead directly induces inhibitory neurotoxicity by blocking the nAChR/channel, whereas NTX may have dual NCB and agonist targets.

Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

KAUST Repository

Qia, Zhi

2010-11-18

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen- defense genes in a Ca 2+-dependent manner.

[99mTc]MAG3-mannosyl-dextran: a receptor-binding radiopharmaceutical for sentinel node detection

International Nuclear Information System (INIS)

Vera, David R.; Wallace, Anne M.; Hoh, Carl K.

2001-01-01

Technetium-99m-labeled benzoyl-mercaptoacetylglycylglycyl-glycine-mannosyl-dextran ([ 99m Tc]MAG 3 -mannosyl-dextran) is a receptor-binding radiotracer that binds to mannose-binding protein, a receptor expressed by reticuloendothelial tissue. This agent is composed of a 10.5-kilodalton molecule of dextran and multiple units of mannose, and benzoyl-mercaptoacetylglycylglycyl-glycine (BzMAG 3 ). The tetraflorophenol-activated ester of BzMAG 3 and the imidate of thiomannose were used to covalently attach BzMAG 3 and mannose to an amino-terminated conjugate of dextran. This yielded a 19-kilodalton macromolecule consisting of 3 BzMAG 3 and 21 mannose units per dextran. Dynamic light scattering was used to measure a mean diameter of 5.5 nanometers for BzMAG 3 -mannosyl-dextran and 0.28 microns for filtered Tc-99m sulfur colloid. A preliminary sentinel node detection study employing right fore and hind footpad injections of [ 99m Tc]MAG 3 -mannosyl-dextran and left fore and hind footpad injections of filtered Tc-99m sulfur colloid demonstrated greater sentinel lymph node uptake by the receptor-binding agent

Preferential Pathway for Glycine Formation in Star-Forming Regions

Science.gov (United States)

Pilling, S.; Boechat-Roberty, H. M.; Baptista, L.; Santos A. C., F.

Interstellar clouds, similar to that from which the solar system was formed, contain many organic molecules including aldehydes, acids, ketones, and sugars Ehrenfreund & Charnley (2000). Those organic compounds have important functions in terrestrial biochemistry and could also have been important in prebiotic synthesis. The simplest amino acid, glycine (NH2CH2COOH), was recently detected in the hot molecular cores Sgr B2(N-LMH), Orion KL, and W51 e1/e2 Kuan et al. (2003). The formic acid (HCOOH) and acetic acid(CH3COOH) have also been detected in those regions Liu et al. (2002), Remijan et al. (2004). The goal of this work is to study experimentally photoionization and photodissociation processes of glycine precursor molecules, acetic acid and formic acid to elucidate a possible preferentially in the glycine synthesis between ice and gas phase. The measurements were taken at the Brazilian Synchrotron Light Laboratory (LNLS), employing soft X-ray photons from a toroidal grating monochromator TGM) beamline (100 - 310 eV). The experimental set up consists of a high vacuum chamber with a Time-Of-Flight Mass Spectrometer (TOF-MS). Mass spectra were obtained using PhotoElectron PhotoIon Coincidence (PEPICO) technique. Kinetic energy distributions and abundances for each ionic fragment have been obtained from the analysis of the corresponding peak shapes in the mass spectra. Dissociative and non-dissociative photoionization cross sections for both molecules were also determined Boechat-Roberty, Pilling & Santos (2005). Due to the high photodissociation cross section of formic acid it is possible that in PDRs regions, just after molecules evaporation from the grains surface, it is almost destructed by soft X-rays, justifying the observed low abundance of HCOOH in gaseous phase Ehrenfreund et al. (2001). Acetic acid have shown to be more stable to the ionizing field, and its main outcomes from dissociation process were the reactive ionic fragments COOH+ and CH3CO+. To

A DFT study of adsorption of glycine onto the surface of BC{sub 2}N nanotube

Energy Technology Data Exchange (ETDEWEB)

Soltani, Alireza, E-mail: Alireza.soltani46@yahoo.com [Joints, Bones and Connective Tissue Research Center, Golestan University of Medical Sciences, Gorgan (Iran, Islamic Republic of); Young Researchers and Elite Club, Gorgan Branch, Islamic Azad University, Gorgan (Iran, Islamic Republic of); Azmoodeh, Zivar [Department of Physics, Payame Noor University, P.O. Box 19395-3697, Tehran (Iran, Islamic Republic of); Javan, Masoud Bezi [Physics Department, Faculty of Sciences, Golestan University, Gorgan (Iran, Islamic Republic of); Lemeski, E. Tazikeh [Department of Chemistry, Gorgan Branch, Islamic Azad University, Gorgan (Iran, Islamic Republic of); Karami, Leila [Institute of Biochemistry and Biophysics, University of Tehran, Tehran (Iran, Islamic Republic of)

2016-10-30

Highlights: • Glycine adsorption over the pristine BC{sub 2}N nanotubes is investigated by DFT calculations. • Adsorption of glycine in its zwitterionic form is stronger in comparison with the radical form. • Adsorption of glycine from its amine head on adsorbent leads to a significant decrease in the electronic properties. - Abstract: A theoretical study of structure and the energy interaction of amino acid glycine (NH{sub 2}CH{sub 2}COOH) with BC{sub 2}N nanotube is crucial for apperception behavior occurring at the nanobiointerface. Herein, we studied the adsorption of glycine in their radical and zwitterionic forms upon the surface of BC{sub 2}N nanotube using M06 functional and 6-311G** standard basis set. We also considered the different orientations of the glycine amino acid on the surface of adsorbent. Further, we found out that the stability of glycine from its carbonyl group is higher than hydroxyl and amine groups. Our results also indicated that the electronic structure of BC{sub 2}N nanotube on the adsorption of glycine from its amine group is more altered than the other groups. Our study exhibits that opto-electronic property of adsorbent is changed after the glycine adsorption.

Effects of glycine on motor performance in rats after traumatic spinal cord injury.

Science.gov (United States)

Gonzalez-Piña, Rigoberto; Nuño-Licona, Alberto

2007-01-01

It has been reported that glycine improves some functions lost after spinal cord injury (SCI). In order to assess the effects of glycine administration on motor performance after SCI, we used fifteen male Wistar rats distributed into three groups: sham (n = 3), spinal-cord injury (n = 6,) and spinal cord injury + glycine (n = 6). Motor performance was assessed using the beam-walking paradigm and footprint analysis. Results showed that for all animals with spinal-cord injury, scores in the beam-walking increased, which is an indication of increased motor deficit. In addition, footprint analysis showed a decrease in stride length and an increase in stride angle, additional indicators of motor deficit. These effects trended towards recovery after 8 weeks of recording and trended toward improvement by glycine administration; the effect was not significant. These results suggest that glycine replacement alone is not sufficient to improve the motor deficits that occur after SCI.

Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels

DEFF Research Database (Denmark)

Mitrovic, Martina; Shahbazian, Anaid; Bock, Elisabeth

2010-01-01

Transient receptor potential ankyrin 1 (TRPA1) channels are expressed by primary afferent neurones and activated by irritant chemicals including allyl isothiocyanate (AITC). Here we investigated whether intracolonic AITC causes afferent input to the spinal cord and whether this response is modifi...

Affinity of hydroxyapatite for furfural and a brown pigment formed by furfural and glycine.

Science.gov (United States)

Nordbö, H; Eriksen, H M; Rölla, G

1979-10-01

The affinity of hydroxyapatite for furfural and a brown pigment formed by furfural and glycine was studied. A series of mixtures containing 1 M furfural and 0.25-2.0 M glycine were incubated at 37 degrees C and aliquots of hydroxyapatite added. The apatite showed a strong affinity for the brown pigment formed, and an excess of glycine in the mixtures appeared to enhance the binding. The adsorption of furfural to hydroxyapatite was estimated by a spectrophotometric method. The data revealed that pretreatment with CaCl2 and glycine significantly increased the adsorption of furfural.

Investigations on the nucleation kinetics of γ-glycine single crystal

International Nuclear Information System (INIS)

Yogambal, C.; Rajan Babu, D.; Ezhil Vizhi, R.

2014-01-01

Single crystals of γ-glycine were grown by slow evaporation technique. The crystalline system was confirmed by single crystal X-ray diffraction analysis. The optical absorption study has shown that the grown crystal possesses lower cut-off wavelength. Solubility and metastable zone width were estimated for different temperatures. The induction period of title compound was determined by varying the temperature and concentration. Nucleation parameters such as Gibbs volume free energy change (ΔG v ), interfacial tension (γ), critical free energy change of the nucleus (ΔG ⁎ ), nucleation rate (J), number of molecules in the critical nucleus (i ⁎ ) have been calculated for the aqueous solution grown γ-glycine single crystals. The second harmonic generation (SHG) of γ-glycine was confirmed by Q-switched Nd:YAG laser technique

Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

Science.gov (United States)

Wang, Liwei; Alzayady, Kamil J; Yule, David I

2016-06-01

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Removal of brownish-black tarnish on silver–copper alloy objects with sodium glycinate

Energy Technology Data Exchange (ETDEWEB)

Cura D’Ars de Figueiredo, João, E-mail: joaoc@ufmg.br; Asevedo, Samara Santos, E-mail: samaranix@hotmail.com; Barbosa, João Henrique Ribeiro, E-mail: joaohrb@yahoo.com.br

2014-10-30

Highlights: • The use of glycinate to remove brownish-black tarnish on silver–copper alloy objects is studied. • The method is easy to use and harmless. It is based in the coordination of Ag and Cu in tarnish with glycinate. • The surface of corroded silver objects and products of reaction were studied and glycinate showed to be very selective for Ag(I) and Cu(II). The selectivity for Ag(I) was studied by means of quantum chemical calculations. - Abstract: This article has the principal aim of presenting a new method of chemical cleaning of tarnished silver–copper alloy objects. The chemical cleaning must be harmless to the health, selective to tarnish removal, and easy to use. Sodium glycinate was selected for the study. The reactions of sodium glycinate with tarnish and the silver–copper alloy were evaluated. Products of the reaction, the lixiviated material, and the esthetics of silver–copper alloy coins (used as prototypes) were studied to evaluate if the proposed method can be applied to the cleaning of silver objects. Silver–copper alloys can be deteriorated through a uniform and superficial corrosion process that produces brownish-black tarnish. This tarnish alters the esthetic of the object. The cleaning of artistic and archeological objects requires more caution than regular cleaning, and it must take into account the procedures for the conservation and restoration of cultural heritage. There are different methods for cleaning silver–copper alloy objects, chemical cleaning is one of them. We studied two chemical cleaning methods that use sodium glycinate and sodium acetylglycinate solutions. Silver–copper alloy coins were artificially corroded in a basic thiourea solution and immersed in solutions of sodium glycinate and sodium acetylglycinate. After immersion, optical microscopy and scanning electron microscopy of the surfaces were studied. The sodium glycinate solution was shown to be very efficient in removing the brownish

Removal of brownish-black tarnish on silver–copper alloy objects with sodium glycinate

International Nuclear Information System (INIS)

Cura D’Ars de Figueiredo, João; Asevedo, Samara Santos; Barbosa, João Henrique Ribeiro

2014-01-01

Highlights: • The use of glycinate to remove brownish-black tarnish on silver–copper alloy objects is studied. • The method is easy to use and harmless. It is based in the coordination of Ag and Cu in tarnish with glycinate. • The surface of corroded silver objects and products of reaction were studied and glycinate showed to be very selective for Ag(I) and Cu(II). The selectivity for Ag(I) was studied by means of quantum chemical calculations. - Abstract: This article has the principal aim of presenting a new method of chemical cleaning of tarnished silver–copper alloy objects. The chemical cleaning must be harmless to the health, selective to tarnish removal, and easy to use. Sodium glycinate was selected for the study. The reactions of sodium glycinate with tarnish and the silver–copper alloy were evaluated. Products of the reaction, the lixiviated material, and the esthetics of silver–copper alloy coins (used as prototypes) were studied to evaluate if the proposed method can be applied to the cleaning of silver objects. Silver–copper alloys can be deteriorated through a uniform and superficial corrosion process that produces brownish-black tarnish. This tarnish alters the esthetic of the object. The cleaning of artistic and archeological objects requires more caution than regular cleaning, and it must take into account the procedures for the conservation and restoration of cultural heritage. There are different methods for cleaning silver–copper alloy objects, chemical cleaning is one of them. We studied two chemical cleaning methods that use sodium glycinate and sodium acetylglycinate solutions. Silver–copper alloy coins were artificially corroded in a basic thiourea solution and immersed in solutions of sodium glycinate and sodium acetylglycinate. After immersion, optical microscopy and scanning electron microscopy of the surfaces were studied. The sodium glycinate solution was shown to be very efficient in removing the brownish

Pore helix domain is critical to camphor sensitivity of transient receptor potential vanilloid 1 channel.

Science.gov (United States)

Marsakova, Lenka; Touska, Filip; Krusek, Jan; Vlachova, Viktorie

2012-04-01

The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.

Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP.

Directory of Open Access Journals (Sweden)

Shlomo S Dellal

Full Text Available Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C(5-maleimide (AM546. Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM or acidic external solution (pH 6.5 elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

International Nuclear Information System (INIS)

Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka; Murakami, Manabu; Ono, Kyoichi; Watanabe, Hiroyuki; Ito, Hiroshi

2013-01-01

Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression

The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity.

Science.gov (United States)

Lund, Trine M; Ploug, Kenneth B; Iversen, Anne; Jensen, Anders A; Jansen-Olesen, Inger

2015-03-01

Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β-hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β-hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release. Energy metabolism and neurotransmission are linked and involve ATP-sensitive potassium (KATP ) channels. However, it is still unclear how and to what degree available energy substrate affects this link. We investigated the effect of changing energy substrate from only glucose to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release. © 2014 International Society for Neurochemistry.

Stereospecific assignments of glycine in proteins by stereospecific deuteration and {sup 15}N labeling

Energy Technology Data Exchange (ETDEWEB)

Hansen, A.P.; Curley, R.W. Jr.; Panigot, M.J.; Fesik, S.W. [Ohio State Univ., Columbus, OH (United States)

1994-12-01

Stereospecific assignments are important for accurately determining the three-dimensional structures of proteins through the use of multidimensional NMR techniques. It is especially important to stereospecifically assign the glycine {alpha}-protons in proteins because of the potential for different backbone conformations of this residue. These stereospecific assignments are critical for interpreting the {sup 3}J{sub NH,{alpha}H} coupling constants and NOEs involving the glycine {alpha}-protons that determine the conformation of this part of the protein. However, it is often difficult to unambiguously obtain the stereospecific assignments for glycine residues by using only NOE data. In this poster, we present a method for unambiguous, stereospecific assignment of the {alpha}-protons of glycine residues. This method involves synthesis of stereo-specifically deuterated and {sup 15}N-labeled Gly using a slightly modified procedure originally described by Woodard and coworkers for the stereoselective deuteration of glycine. The stereospecifically deuterated and {sup 15}N-labeled Gy has been incorporated into recombinant proteins expressed in both bacterial systems (FKBP) and mammalian cells (u-PA). Two- and three-dimensional isotope-filtered and isotope-edited NMR experiments were used to obtain the stereospecific assignments of the glycine {alpha}-protons for these proteins.

Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

Science.gov (United States)

Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

2017-01-01

The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

Interactions of Heterodera glycines, Macrophomina phaseolina, and Mycorrhizal Fungi on Soybean in Kansas.

Science.gov (United States)

Winkler, H E; Hetrick, B A; Todd, T C

1994-12-01

The impact of naturally occurring arbuscular mycorrhizal fungi on soybean growth and their interaction with Heterodera glycines were evaluated in nematode-infested and uninfested fields in Kansas. Ten soybean cultivars from Maturity Groups III-V with differential susceptibility to H. glycines were treated with the fungicide benomyl to suppress colonization by naturally occurring mycorrhizal fungi and compared with untreated control plots. In H. glycines-infested soil, susceptible cultivars exhibited 39% lower yields, 28% lower colonization by mycorrhizal fungi, and an eightfold increase in colonization by the charcoal rot fungus, Macrophomina phaseolina, compared with resistant cultivars. In the absence of the nematode, susceptible cultivars exhibited 10% lower yields than resistant cultivars, root colonization of resistant vs. susceptible soybean by mycorrhizal fungi varied with sampling date, and there were no differences in colonization by M. phaseolina between resistant and susceptible cultivars. Benomyl application resulted in 19% greater root growth and 9% higher seed yields in H. glycines-infested soil, but did not affect soybean growth and yield in the absence of the nematode. Colonization of soybean roots by mycorrhizal fungi was negatively correlated with H. glycines population densities due to nematode antagonism to the mycorrhizal fungi rather than suppression of nematode populations. Soybean yields were a function of the pathogenic effects of H. glycines and M. phaseolina, and, to a lesser degree, the stimulatory effects of mycorrhizal fungi.

Hydration properties of mechanosensitive channel pores define the energetics of gating

International Nuclear Information System (INIS)

Anishkin, A; Akitake, B; Kamaraju, K; Chiang, C-S; Sukharev, S

2010-01-01

Opening of ion channels directly by tension in the surrounding membrane appears to be the most ancient and simple mechanism of gating. Bacterial mechanosensitive channels MscL and MscS are the best-studied tension-gated nanopores, yet the key physical factors that define their gating are still hotly debated. Here we present estimations, simulations and experimental results showing that hydration of the pore might be one of the major parameters defining the thermodynamics and kinetics of mechanosensitive channel gating. We associate closing of channel pores with complete dehydration of the hydrophobic gate (occlusion by 'vapor lock') and formation of two water-vapor interfaces above and below the constriction. The opening path is the expansion of these interfaces, ultimately leading to wetting of the hydrophobic pore, which does not appear to be the exact reverse of the closing path, thus producing hysteresis. We discuss specifically the role of polar groups (glycines) buried in narrow closed conformations but exposed in the open states that change the wetting characteristics of the pore lining and stabilize conductive states of the channels.

Beta amyloid differently modulate nicotinic and muscarinic receptor subtypes which regulate in vitro and in vivo the release of glycine in the rat hippocampus

Directory of Open Access Journals (Sweden)

Stefania eZappettini

2012-07-01

Full Text Available Using both in vitro (hippocampal synaptosomes in superfusion and in vivo (microdialysis approaches we investigated whether and to what extent β amyloid peptide 1-40 (Aβ 1-40 interferes with the cholinergic modulation of the release of glycine (GLY in the rat hippocampus. The nicotine-evoked overflow of endogenous GLY in hippocampal synaptosomes in superfusion was significantly inhibited by Aβ 1-40 (10 nM while increasing the concentration to 100 nM the inhibitory effect did not further increase. Both the Choline (Ch (α7 agonist; 1 mM and the 5-Iodo-A-85380 dihydrochloride (5IA85380, α4β2 agonist; 10 nM-evoked GLY overflow were inhibited by Aβ1-40 at 100 nM but not at 10nM concentrations. The KCl evoked [3H]GLY and [3H]Acetylcholine (ACh overflow were strongly inhibited in presence of oxotremorine; however this inhibitory muscarinic effect was not affected by Aβ1-40. The effects of Aβ1-40 on the administration of nicotine, veratridine, 5IA85380 and PHA 543613 hydrochloride (PHA543613 (a selective agonist of α7 subtypes on hippocampal endogenous GLY release in vivo were also studied. Aβ 1-40 significantly reduced (at 10 μM but not at 1 μM the nicotine evoked in vivo release of GLY. Aβ 1-40 (at 10 μM but not at 1 μM significantly inhibited the PHA543613 (1 mM-elicited GLY overflow while was ineffective on the GLY overflow evoked by 5IA85380 (1 mM. Aβ 40-1 (10 μM did not produce any inhibitory effect on nicotine evoked GLY overflow both in the in vitro and in vivo experiments. Our results indicate that a the cholinergic modulation of the release of GLY occurs by the activation of both α7 and α4β2 nicotinic ACh receptors (nAChRs as well as by the activation of inhibitory muscarinic ACh receptors (mAChRs and b Aβ 1-40 can modulate cholinergic evoked GLY release exclusively through the interaction with α7 and the α4β2 nAChR nicotinic receptors but not through mAChR subtypes.

Imaging the PCP site of the NMDA ion channel

Energy Technology Data Exchange (ETDEWEB)

Waterhouse, Rikki N. E-mail: rnw7@columbia.edu

2003-11-01

The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed.

Imaging the PCP site of the NMDA ion channel

International Nuclear Information System (INIS)

Waterhouse, Rikki N.

2003-01-01

The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed

Cyclophosphamide-induced cystitis reduces ASIC channel but enhances TRPV1 receptor function in rat bladder sensory neurons.

Science.gov (United States)

Dang, Khoa; Bielefeldt, Klaus; Gebhart, G F

2013-07-01

Using patch-clamp techniques, we studied the plasticity of acid-sensing ion channels (ASIC) and transient receptor potential V1 (TRPV1) channel function in dorsal root ganglia (DRG) neurons retrogradely labeled from the bladder. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally on days 1, 3, and 5. On day 6, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T13-L2) DRG were removed and dissociated. Bladders and bladder DRG neurons from CYP-treated rats showed signs of inflammation (greater myeloperoxidase activity; lower intramuscular wall pH) and increased size (whole cell capacitance), respectively, compared with controls. Most bladder neurons (>90%) responded to protons and capsaicin. Protons produced multiphasic currents with distinct kinetics, whereas capsaicin always triggered a sustained response. The TRPV1 receptor antagonist A-425619 abolished capsaicin-triggered currents and raised the threshold of heat-activated currents. Prolonged exposure to an acidic environment (pH range: 7.2 to 6.6) inhibited proton-evoked currents, potentiated the capsaicin-evoked current, and reduced the threshold of heat-activated currents in LS and TL bladder neurons. CYP treatment reduced density but not kinetics of all current components triggered by pH 5. In contrast, CYP-treatment was associated with an increased current density in response to capsaicin in LS and TL bladder neurons. Correspondingly, heat triggered current at a significantly lower temperature in bladder neurons from CYP-treated rats compared with controls. These results reveal that cystitis differentially affects TRPV1- and ASIC-mediated currents in both bladder sensory pathways. Acidification of the bladder wall during inflammation may contribute to changes in nociceptive transmission mediated through the TRPV1 receptor, suggesting a role for TRPV1 in hypersensitivity associated with cystitis.

In vivo evaluation of [11C]-3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole- 2-carboxylic acid ([11C]3MPICA) as a PET radiotracer for the glycine site of the NMDA ion channel

International Nuclear Information System (INIS)

Waterhouse, Rikki N.; Sultana, Abida; Laruelle, M.

2002-01-01

Alterations in normal NMDA receptor composition, densities and function have been implicated in the pathophysiology of certain neurological and neuropsychiatric disorders such as Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. In our first effort to provide PET ligands for the NMDA/glycine site, we reported the synthesis of a novel high affinity glycine site ligand, 3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2 -carboxylic acid ((3MPICA), Ki=4.8±0.9 nM) and the corresponding carbon-11 labeled PET ligand, [ 11 C]3MPICA. We report here the in vivo evaluation of [ 11 C]3MPICA in rats. Biodistribution analysis revealed that [ 11 C]3MPICA exhibited low degree of brain penetration and high blood concentration. The average uptake at two minutes was highest in the cerebellum (0.19±0.04 %ID/g) and thalamus (0.18±0.05 %ID/g) and lower in the hippocampus (0.13±0.03) and frontal cortex (0.11±0.04 %ID/g). The radioactivity cleared quickly from all brain regions examined. Administration of unlabeled 3MPICA (1 mg/kg, i.v.) revealed at 60 minutes a small general reduction in regional brain radioactivity concentrations in treated animals versus controls, however, the blood radioactivity concentration was also lowered, confounding the assessment of the degree of saturable binding. Warfarin co-administration (100 mg/kg, i.v.) significantly lowered blood activity at 5 minutes post-injection (-27%, P 11 C]3MPICA does not appear to be a promising PET radiotracer for in vivo use

Pannexin-1 channels in epilepsy.

Science.gov (United States)

Aquilino, Mark S; Whyte-Fagundes, Paige; Zoidl, Georg; Carlen, Peter L

2017-09-05

Pannexin-1 (Panx1) expression is raised in several animal seizure models and in resected human epileptic brain tissue, suggesting relevance to epilepsy. Multiple factors that are characteristic of seizures are thought to regulate Panx1 channel opening, including elevated levels of extracellular K + . Panx1, when open, 1) releases ATP, glutamate, and other metabolites into the extracellular medium, and 2) may depolarize the membrane due to a channel reversal potential around 0mV. Resultant ATP release from stimulated Panx1 can activate purinergic receptors, including P2X7 receptors. Glutamate and other signaling molecules released by Panx1 opening may have both excitatory and inhibitory actions on seizure generation. This review examines the critical and complex roles of Panx1 channels in epilepsy, which could provide a basis for future therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

Science.gov (United States)

McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

2014-01-01

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Exogenous Glycine Nitrogen Enhances Accumulation of Glycosylated Flavonoids and Antioxidant Activity in Lettuce (Lactuca sativa L.

Directory of Open Access Journals (Sweden)

Xiao Yang

2017-12-01

Full Text Available Glycine, the simplest amino acid in nature and one of the most abundant free amino acids in soil, is regarded as a model nutrient in organic nitrogen studies. To date, many studies have focused on the uptake, metabolism and distribution of organic nitrogen in plants, but few have investigated the nutritional performance of plants supplied with organic nitrogen. Lettuce (Lactuca sativa L., one of the most widely consumed leafy vegetables worldwide, is a significant source of antioxidants and bioactive compounds such as polyphenols, ascorbic acid and tocopherols. In this study, two lettuce cultivars, Shenxuan 1 and Lollo Rossa, were hydroponically cultured in media containing 4.5, 9, or 18 mM glycine or 9 mM nitrate (control for 4 weeks, and the levels of health-promoting compounds and antioxidant activity of the lettuce leaf extracts were evaluated. Glycine significantly reduced fresh weight compared to control lettuce, while 9 mM glycine significantly increased fresh weight compared to 4.5 or 18 mM glycine. Compared to controls, glycine (18 mM for Shenxuan 1; 9 mM for Lollo Rossa significantly increased the levels of most antioxidants (including total polyphenols, α-tocopherol and antioxidant activity, suggesting appropriate glycine supply promotes antioxidant accumulation and activity. Glycine induced most glycosylated quercetin derivatives and luteolin derivatives detected and decreased some phenolic acids compared to nitrate treatment. This study indicates exogenous glycine supplementation could be used strategically to promote the accumulation of health-promoting compounds and antioxidant activity of hydroponically grown lettuce, which could potentially improve human nutrition.

Exogenous Glycine Nitrogen Enhances Accumulation of Glycosylated Flavonoids and Antioxidant Activity in Lettuce (Lactuca sativa L.).

Science.gov (United States)

Yang, Xiao; Cui, Xiaoxian; Zhao, Li; Guo, Doudou; Feng, Lei; Wei, Shiwei; Zhao, Chao; Huang, Danfeng

2017-01-01

Glycine, the simplest amino acid in nature and one of the most abundant free amino acids in soil, is regarded as a model nutrient in organic nitrogen studies. To date, many studies have focused on the uptake, metabolism and distribution of organic nitrogen in plants, but few have investigated the nutritional performance of plants supplied with organic nitrogen. Lettuce ( Lactuca sativa L.), one of the most widely consumed leafy vegetables worldwide, is a significant source of antioxidants and bioactive compounds such as polyphenols, ascorbic acid and tocopherols. In this study, two lettuce cultivars, Shenxuan 1 and Lollo Rossa, were hydroponically cultured in media containing 4.5, 9, or 18 mM glycine or 9 mM nitrate (control) for 4 weeks, and the levels of health-promoting compounds and antioxidant activity of the lettuce leaf extracts were evaluated. Glycine significantly reduced fresh weight compared to control lettuce, while 9 mM glycine significantly increased fresh weight compared to 4.5 or 18 mM glycine. Compared to controls, glycine (18 mM for Shenxuan 1; 9 mM for Lollo Rossa) significantly increased the levels of most antioxidants (including total polyphenols, α-tocopherol) and antioxidant activity, suggesting appropriate glycine supply promotes antioxidant accumulation and activity. Glycine induced most glycosylated quercetin derivatives and luteolin derivatives detected and decreased some phenolic acids compared to nitrate treatment. This study indicates exogenous glycine supplementation could be used strategically to promote the accumulation of health-promoting compounds and antioxidant activity of hydroponically grown lettuce, which could potentially improve human nutrition.

Biological control of Heterodera glycines by spore-forming plant growth-promoting rhizobacteria (PGPR on soybean.

Directory of Open Access Journals (Sweden)

Ni Xiang

Full Text Available Heterodera glycines, the soybean cyst nematode, is the most economically important plant-parasitic nematode on soybean production in the U.S. The objectives of this study were to evaluate the potential of plant growth-promoting rhizobacteria (PGPR strains for mortality of H. glycines J2 in vitro and for reducing nematode population density on soybean in greenhouse, microplot, and field trials. The major group causing mortality to H. glycines in vitro was the genus Bacillus that consisted of 92.6% of the total 663 PGPR strains evaluated. The subsequent greenhouse, microplot, and field trials indicated that B. velezensis strain Bve2 consistently reduced H. glycines cyst population density at 60 DAP. Bacillus mojavensis strain Bmo3 suppressed H. glycines cyst and total H. glycines population density under greenhouse conditions. Bacillus safensis strain Bsa27 and Mixture 1 (Bve2 + Bal13 reduced H. glycines cyst population density at 60 DAP in the field trials. Bacillus subtilis subsp. subtilis strains Bsssu2 and Bsssu3, and B. velezensis strain Bve12 increased early soybean growth including plant height and plant biomass in the greenhouse trials. Bacillus altitudinis strain Bal13 increased early plant growth on soybean in the greenhouse and microplot trials. Mixture 2 (Abamectin + Bve2 + Bal13 increased early plant growth in the microplot trials at 60 DAP, and also enhanced soybean yield at harvest in the field trials. These results demonstrated that individual PGPR strains and mixtures can reduce H. glycines population density in the greenhouse, microplot, and field conditions, and increased yield of soybean.

Biological control of Heterodera glycines by spore-forming plant growth-promoting rhizobacteria (PGPR) on soybean.

Science.gov (United States)

Xiang, Ni; Lawrence, Kathy S; Kloepper, Joseph W; Donald, Patricia A; McInroy, John A

2017-01-01

Heterodera glycines, the soybean cyst nematode, is the most economically important plant-parasitic nematode on soybean production in the U.S. The objectives of this study were to evaluate the potential of plant growth-promoting rhizobacteria (PGPR) strains for mortality of H. glycines J2 in vitro and for reducing nematode population density on soybean in greenhouse, microplot, and field trials. The major group causing mortality to H. glycines in vitro was the genus Bacillus that consisted of 92.6% of the total 663 PGPR strains evaluated. The subsequent greenhouse, microplot, and field trials indicated that B. velezensis strain Bve2 consistently reduced H. glycines cyst population density at 60 DAP. Bacillus mojavensis strain Bmo3 suppressed H. glycines cyst and total H. glycines population density under greenhouse conditions. Bacillus safensis strain Bsa27 and Mixture 1 (Bve2 + Bal13) reduced H. glycines cyst population density at 60 DAP in the field trials. Bacillus subtilis subsp. subtilis strains Bsssu2 and Bsssu3, and B. velezensis strain Bve12 increased early soybean growth including plant height and plant biomass in the greenhouse trials. Bacillus altitudinis strain Bal13 increased early plant growth on soybean in the greenhouse and microplot trials. Mixture 2 (Abamectin + Bve2 + Bal13) increased early plant growth in the microplot trials at 60 DAP, and also enhanced soybean yield at harvest in the field trials. These results demonstrated that individual PGPR strains and mixtures can reduce H. glycines population density in the greenhouse, microplot, and field conditions, and increased yield of soybean.

Direct proton conductance through the TRPV1 pore and multimerization of TRPV channel subunits

OpenAIRE

Hellwig, Nicole Barbara

2010-01-01

TRPV1-induced intracellular acidification: The vanilloid receptor-related transient receptor potential channels (TRPV) belong to the superfamily of hexahelical cation channels and are integral components of thermosensation, pain perception and Ca2+-reabsorption in kidney and intestine. The vanilloid receptor (TRPV1), a poorly selective cation channel, is expressed in dorsal root ganglion (DRG) neurons and is regulated by diverse stimuli including capsaicin, endovanilloids and heat. Since a...

First report of the Soybean Cyst Nematode, Heterodera glycines, in New York

Science.gov (United States)

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the most damaging pathogen of soybean (Glycine max (L.) Merr.), causing more than $1 billion in yield losses annually in the United States (Koenning and Wrather 2010). The SCN distribution map updated in 2014 showed that SCN were dete...

The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

Science.gov (United States)

Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

2016-01-01

Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

DEFF Research Database (Denmark)

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus

2017-01-01

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D...

Pharmacological Conversion of a Cardiac Inward Rectifier into an Outward Rectifier Potassium Channel.

Science.gov (United States)

Moreno-Galindo, Eloy G; Sanchez-Chapula, Jose A; Tristani-Firouzi, Martin; Navarro-Polanco, Ricardo A

2016-09-01

Potassium (K(+)) channels are crucial for determining the shape, duration, and frequency of action-potential firing in excitable cells. Broadly speaking, K(+) channels can be classified based on whether their macroscopic current outwardly or inwardly rectifies, whereby rectification refers to a change in conductance with voltage. Outwardly rectifying K(+) channels conduct greater current at depolarized membrane potentials, whereas inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward currents through inwardly rectifying K(+) channels are reduced at more depolarized potentials. However, the acetylcholine-gated K(+) channel (KACh) conducts current that inwardly rectifies when activated by some ligands (such as acetylcholine), and yet conducts current that outwardly rectifies when activated by other ligands (for example, pilocarpine and choline). The perplexing and paradoxical behavior of KACh channels is due to the intrinsic voltage sensitivity of the receptor that activates KACh channels, the M2 muscarinic receptor (M2R). Emerging evidence reveals that the affinity of M2R for distinct ligands varies in a voltage-dependent and ligand-specific manner. These intrinsic receptor properties determine whether current conducted by KACh channels inwardly or outwardly rectifies. This review summarizes the most recent concepts regarding the intrinsic voltage sensitivity of muscarinic receptors and the consequences of this intriguing behavior on cardiac physiology and pharmacology of KACh channels. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

Science.gov (United States)

Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

2016-11-01

Inward rectifier K + channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP 2 ). Stimulation of the Ca 2+ -sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both G q/11 , which decreases PIP 2 , and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP 2 . How membrane PIP 2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K + are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K + current (I K1 ) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP 2 reporter tubby-R332H-cYFP to monitor PIP 2 levels, we found that CaR activation in HEK293T cells increased membrane PIP 2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP 2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K + channels by accelerating PIP 2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

Science.gov (United States)

Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

2016-05-01

Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

Transcranial Random Noise Stimulation-induced plasticity is NMDA-receptor independent but sodium-channel blocker and benzodiazepines sensitive

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Leila eChaieb

2015-04-01

Full Text Available Background: Application of transcranial random noise stimulation (tRNS between 0.1 and 640 Hz of the primary motor cortex (M1 for 10 minutes induces a persistent excitability increase lasting for at least 60 minutes. However, the mechanism of tRNS-induced cortical excitability alterations is not yet fully understood. Objective: The main aim of this study was to get first efficacy data with regard to the possible neuronal effect of tRNS. Methods: Single-pulse transcranial magnetic stimulation (TMS was used to measure levels of cortical excitability before and after combined application of tRNS at an intensity of 1mA for 10mins stimulation duration and a pharmacological agent (or sham on 8 healthy male participants. Results: The sodium channel blocker carbamazepine showed a tendency towards inhibiting MEPs 5-60 mins poststimulation. The GABAA agonist lorazepam suppressed tRNS-induced cortical excitability increases at 0-20 and 60 min time points. The partial NMDA receptor agonist D-cycloserine, the NMDA receptor antagonist dextromethorphan and the D2/D3 receptor agonist ropinirole had no significant effects on the excitability increases seen with tRNS.Conclusions: In contrast to transcranial direct current stimulation (tDCS, aftereffects of tRNS are seem to be not NMDA receptor dependent and can be suppressed by benzodiazepines suggesting that tDCS and tRNS depend upon different mechanisms.

Somatic embryogenesis in cell cultures of Glycine species.

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Gamborg, O L; Davis, B P; Stahlhut, R W

1983-08-01

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

Interaction of H2S with Calcium Permeable Channels and Transporters

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Weihua Zhang

2015-01-01

Full Text Available A growing amount of evidence has suggested that hydrogen sulfide (H2S, as a gasotransmitter, is involved in intensive physiological and pathological processes. More and more research groups have found that H2S mediates diverse cellular biological functions related to regulating intracellular calcium concentration. These groups have demonstrated the reciprocal interaction between H2S and calcium ion channels and transporters, such as L-type calcium channels (LTCC, T-type calcium channels (TTCC, sodium/calcium exchangers (NCX, transient receptor potential (TRP channels, β-adrenergic receptors, and N-methyl-D-aspartate receptors (NMDAR in different cells. However, the understanding of the molecular targets and mechanisms is incomplete. Recently, some research groups demonstrated that H2S modulates the activity of calcium ion channels through protein S-sulfhydration and polysulfide reactions. In this review, we elucidate that H2S controls intracellular calcium homeostasis and the underlying mechanisms.

TAURINE REGULATION OF VOLTAGE-GATED CHANNELS IN RETINAL NEURONS

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Rowan, Matthew JM; Bulley, Simon; Purpura, Lauren; Ripps, Harris; Shen, Wen

2017-01-01

Taurine activates not only Cl−-permeable ionotropic receptors, but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl−-permeable receptors with an inhibitory “cocktail” consisting of picrotoxin, SR95531, and strychnine. We found that taurine’s metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K+ channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca2+ channels in retinal neurons, which were insensitive to the potent GABAB receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain. PMID:23392926

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

International Nuclear Information System (INIS)

Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo

2005-01-01

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology

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Smith, Amy C.; Hristov, Kiril L.; Cheng, Qiuping; Xin, Wenkuan; Parajuli, Shankar P.; Earley, Scott; Malysz, John

2013-01-01

Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1–30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1–7 μM and 70–80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5–50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling. PMID:23302778

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

in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis......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...

Resolving the limitations of using glycine as EPR dosimeter in the intermediate level of gamma dose

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Aboelezz, E.; Hassan, G. M.

2018-04-01

The dosimetric properties of the simplest amino acid "glycine"- using EPR technique- were investigated in comparison to reference standard alanine dosimeter. The EPR spectrum of glycine at room temperature is complex, but immediately after irradiation, it appears as a triplet hyperfine structure probably due to the dominant contribution of the (•CH2COO-) radical. The dosimetric peak of glycine is at g-factor 2.0026 ± 0.0015 and its line width is 9 G at large modulation amplitude (7 G). The optimum microwave was studied and was found to be as alanine 8 mW; the post-irradiation as well as the dose rate effects were discussed. Dosimetric peak intensity of glycine fades rapidly to be about one quarter of its original value during 20 days for dried samples and it stabilizes after that. The dose response study in an intermediate range (2-1000 Gy) reveals that the glycine SNR is about 2 times more than that of alanine pellets when measured immediately after irradiation and 4 times more than that of glycine itself after 22 days of irradiation. The effect of energy dependence was studied and interpreted theoretically by calculation of mass energy absorption coefficient. The calculated combined uncertainties for glycine and alanine are nearly the same and were found to be 2.42% and 2.33%, respectively. Glycine shows interesting dosimetric properties in the range of ionizing radiation doses investigated.

Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex.

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Crook, J; Hendrickson, A; Robinson, F R

2006-09-15

Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density

A structural view of ligand-dependent activation in thermoTRP channels

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Ximena eSteinberg

2014-05-01

Full Text Available Transient Receptor Potential (TRP proteins are a large family of ion channels, grouped intoseven sub-families. Although great advances have been made regarding the activation andmodulation of TRP channel activity, detailed molecular mechanisms governing TRPchannel gating are still needed. Sensitive to electric, chemical, mechanical, and thermalcues, TRP channels are tightly associated with the detection and integration of sensoryinput, emerging as a model to study the polymodal activation of ion channel proteins.Among TRP channels, the temperature-activated kind constitute a subgroup by itself,formed by Vanilloid receptors 1-4, Melastatin receptors 2, 4, 5 and 8, TRPC5, and TRPA1.Some of the so-called thermoTRP channels participate in the detection of noxious stimulimaking them an interesting pharmacological target for the treatment of pain. However, thepoor specificity of the compounds available in the market represents an important obstacleto overcome. Understanding the molecular mechanics underlying ligand-dependentmodulation of TRP channels may help with the rational design of novel syntheticanalgesics. The present review focuses on the structural basis of ligand-dependentactivation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection ofligand-binding sites within TRPV1, PIP 2 -dependent modulation of TRP channels, and thestructure of natural and synthetic ligands.

Synthesis of Nb-doped SrTiO₃ by a modified glycine-nitrate process

DEFF Research Database (Denmark)

Blennow Tullmar, Peter; Kammer Hansen, Kent; Wallenberg, L.R.

2007-01-01

The objective of the present investigation was to develop a technique to synthesize submicronic particles of Nb-doped strontium titanate with a homogeneous composition. This was achieved by a modified glycine-nitrate process, using Ti-lactate, Nb-oxalate, and Sr(NO3)(2) as starting materials....... A combination of both citric acid and glycine was needed in order to integrate the useful features of both complexation and combustion natures of citric acid and glycine, respectively. The amount of citric acid, glycine, and nitrates in the starting solution, as well as the source for extra nitrates...

Effect of amount of glycine as fuel in obtaining nanocomposite Ni/NiO

International Nuclear Information System (INIS)

Simoes, A.N.; Simoes, V.N.; Neiva, L.S.; Quirino, M.R.; Vieira, D.A.; Gama, L.

2010-01-01

This paper proposes to investigate the effect of the amount of glycine in obtaining nanocomposite Ni/NiO synthesized by combustion reaction technique. The amount of glycine used was calculated on the stoichiometric composition of 50% and 100%. Characterizations by X-ray diffraction (XRD), N2 adsorption by the BET method and scanning electron microscopy (SEM) were performed with powder of Ni/NiO result. The analysis of X-ray diffraction showed the presence of crystalline NiO phase in the presence of nickel as a secondary phase, whose amount increased with the amount of glycine. Increasing the concentration of glycine also caused an increase in surface area, which ranged from 1.1 to 1.4 m 2 /g. The micrographs revealed the formation of soft agglomerates with porous appearance and easy dispersions. It can be concluded that the synthesis is effective to obtain nanosized powders. (author)

A simple and highly selective 2,2-diferrocenylpropane-based multi-channel ion pair receptor for Pb(2+) and HSO4(-).

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Wan, Qian; Zhuo, Ji-Bin; Wang, Xiao-Xue; Lin, Cai-Xia; Yuan, Yao-Feng

2015-03-28

A structurally simple, 2,2-diferrocenylpropane-based ion pair receptor 1 was synthesized and characterized by (1)H NMR, (13)C NMR, HRMS, elemental analyses, and single-crystal X-ray diffraction. The ion pair receptor 1 showed excellent selectivity and sensitivity towards Pb(2+) with multi-channel responses: a fluorescence enhancement (more than 42-fold), a notable color change from yellow to red, redox anodic shift (ΔE1/2 = 151 mV), while HSO4(-) promoted fluorescence enhancement when Pb(2+) or Zn(2+) was bonded to the cation binding-site. (1)H NMR titration and density functional theory were performed to reveal the sensing mechanism based on photo-induced electron transfer (PET).

Metabolism of L-leucine-U-14C in young rats fed excess glycine diets

International Nuclear Information System (INIS)

Takeuchi, Hisanao; Tadauchi, Nobuo; Muramatsu, Keiichiro

1975-01-01

As reported previously, while the growth-depressing effect of excess glycine was prevented by supplementing L-arginine and L-methionine, the degradation of glycine-U-(SUP 14)C into expired carbon dioxide was not accelerated by the supplement of both amino acids. However, it was found that the incorporation of the isotope into the lipids of livers and carcasses increased in the rats fed the excess glycine diet containing both amino acids. The lipid synthesis utilizing excess glycine may be accelerated by adding both amino acids to the 10% casein diet containing excess glycine. In the present experiment, the metabolic fate of L-leucine-U-(SUP 14)C was studied with the rats fed the excess glycine diet with or without L-arginine and L-methionine. 10% casein (10C), 10% casein diet containing 7% glycine (10C7G), or 10C7G Supplemented with 1.4% L-arginine-HCL and 0.9% L-methionine (10C7GArgMet) was fed to each rat, and the diet suspension containing 4 sup(μ)Ci of L-leucine-U-(SUP 14)C per 100 g of body weight was fed forcibly after 12 hr fast. The radioactivity in expired carbon dioxide, TCA soluble fraction, protein, glycogen, lipids and urine, and the concentration of free amino acids in blood plasma, livers and urine were measured. The body weight gain and food intake of the 10C7G group were much smaller than those of the other groups. The recovery of (SUP 14)C-radioactivity in expired carbon dioxide was much lower in the 10C7GArgMet group than that of the other groups. (Kako, I.)

Novel transcripts of the estrogen receptor α gene in channel catfish

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Patino, Reynaldo; Xia, Zhenfang; Gale, William L.; Wu, Chunfa; Maule, Alec G.; Chang, Xiaotian

2000-01-01

Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERα cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERα variants of different sizes. Relative to the catfish ERα (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERα (36 residues longer) and Short-ERα (389 residues shorter). The 5′-end of Long-ERα cDNA is identical to that of Medium-ERα but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERα binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ERα but lower than reported for catfish ERβ. Short-ERα cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERα RNAs that include the size range of the ERα cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERα cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERα antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERα antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERα mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may

Thermodynamics of proton dissociations from aqueous glycine at temperatures from 278.15 to 393.15 K, molalities from 0 to 1.0 mol . kg-1, and at the pressure 0.35 MPa: Apparent molar heat capacities and apparent molar volumes of glycine, glycinium chloride, and sodium glycinate

International Nuclear Information System (INIS)

Ziemer, S.P.; Niederhauser, T.L.; Merkley, E.D.; Price, J.L.; Sorenson, E.C.; McRae, B.R.; Patterson, B.A.; Origlia-Luster, M.L.; Woolley, E.M.

2006-01-01

We have measured the densities of aqueous solutions of glycine, glycine plus equimolal HCl, and glycine plus equimolal NaOH at temperatures 278.15 ≤ T/K ≤ 368.15, molalities 0.01 ≤ m/mol . kg -1 ≤ 1.0, and at p = 0.35 MPa, using a vibrating tube densimeter. We have also measured the heat capacities of these solutions at 278.15 ≤ T/K ≤ 393.15 and at the same m and p using a fixed-cell differential scanning calorimeter. We used the densities to calculate apparent molar volumes V φ and the heat capacities to calculate apparent molar heat capacities C p,φ for these solutions. We used our results and values of V φ (T, m) and C p,φ (T, m) for HCl(aq), NaOH(aq), NaCl(aq) from the literature to calculate parameters for Δ r C p,m (T, m) for the first and second proton dissociations from protonated aqueous cationic glycine. We then integrated this value of Δ r C p,m (T, m) in an iterative algorithm, using Young's Rule to account for the effects of speciation and chemical relaxation on the observed V φ and C p,φ of the solutions. This procedure yielded parameters for V φ (T, m) and C p,φ (T, m) for glycinium chloride {H 2 Gly + Cl - (aq)} and sodium glycinate {Na + Gly - (aq)} which successfully modeled our observed results. We have then calculated values of Δ r C p,m , Δ r H m , Δ r V m , and pQ a for the first and second proton dissociations from protonated aqueous glycine as functions of T and m

THE INCORPORATION OF RADIOACTIVITY FROM GLYCINE-C$sup 14$ BY MAMMALIAN SPERMATOZOA

Energy Technology Data Exchange (ETDEWEB)

Graves, C. N.

1962-05-15

The metabolic pathways of glycine incorporation were investigated by biochemical and radibautographic methods. Results show that glycine is utilized hy bovine spermatoza and is incorporated into all fractions of the sperm cell. Incorporation into the nucleic acid fraction and especially into thymine indicates that there is a turnover in the desoxyribenucleic acid during storage of bovine spermatoza. (C.H.)

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.

Voltage-gated sodium channels in taste bud cells.

Science.gov (United States)

Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

2009-03-12

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. 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. 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.

Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

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Gelband, C H; Sumners, C; Lu, D; Raizada, M K

1998-02-27

The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8.

Science.gov (United States)

De Petrocellis, Luciano; Vellani, Vittorio; Schiano-Moriello, Aniello; Marini, Pietro; Magherini, Pier Cosimo; Orlando, Pierangelo; Di Marzo, Vincenzo

2008-06-01

The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Delta(9)-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC(50) = 60 nM) and the least potent being CBG and CBD acid (EC(50) = 3.4-12.0 microM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC(50) = 34.3 microM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC(50) = 70-160 nM), whereas CBD acid was the least potent compound (IC(50) = 0.9-1.6 microM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC(50) = 4.5 microM) similar to that of anandamide (IC(50) = 10 microM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

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Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

2011-01-01

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal

Design and synthesis of enantiomerically enriched, radiolabeled MK-801 analogs as potential radiotracers for imaging and autoradiographic studies of the NMDA receptor-ion channel complex

International Nuclear Information System (INIS)

Eng, W.S.; Burns, H.D.; Gibson, R.E.; Ransom, R.W.; Thorpe, H.; Fioravanit, C.; Britcher, S.F.; Magill, C.A.; Solomon, H.F.; Dannals, R.F.; Wilson, A.A.; Ravert, H.T.; Wagner, H.N.

1989-01-01

MK-801 is a potent, non-competitive antagonist for the N-methyl-D-asspartate (NMDA) receptor-ion channel complex. This complex is though to be involved in nerve cell damage in stroke patients when excess calcium is released through the activated channel. A thorough understanding of drug interactions with the NMDA receptor complex could lead to improved therapy for reducing hypoxic-ischemic neuronal injuries in stroke patients. Based on the results of extensive structure-activity studies, the authors have developed several enantiomerically enriched, radiolabeled analogs of MK-801, including: 3-1231-MK-801 for Single Photon Emission Computed tomography (SPECT); 3-1251-MK-801 for in-vivo and in-vitro autoradiography; 8-11C-MeO-MK-801 for Positron Emission Tomography (PET). Details of the synthesis of these radiotracers and their application to both in-vitro and in-vivo studies are described

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

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Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Involvement of plasma membrane Ca2+ channels, IP3 receptors, and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct.

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Tamashiro, Hirotake; Yoshino, Masami

2014-12-01

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29±0.009 Hz (n=43) and an amplitude of 14.6±1.25 mg (n=29). SRCs completely disappeared following removal of extracellular Ca2+ using a solution containing 5mM EGTA. Application of the non-specific Ca2+ channel blockers Co2+, Ni2+, and Cd2+ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca2+ entry through plasma membrane Ca2+ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca2+ by locking ryanodine receptor (RyR)-Ca2+ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30-50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP3R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca2+-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca2+ influx through plasma membrane Ca2

The signaling role for chloride in the bidirectional communication between neurons and astrocytes.

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Wilson, Corinne S; Mongin, Alexander A

2018-01-09

It is well known that the electrical signaling in neuronal networks is modulated by chloride (Cl - ) fluxes via the inhibitory GABA A and glycine receptors. Here, we discuss the putative contribution of Cl - fluxes and intracellular Cl - to other forms of information transfer in the CNS, namely the bidirectional communication between neurons and astrocytes. The manuscript (i) summarizes the generic functions of Cl - in cellular physiology, (ii) recaps molecular identities and properties of Cl - transporters and channels in neurons and astrocytes, and (iii) analyzes emerging studies implicating Cl - in the modulation of neuroglial communication. The existing literature suggests that neurons can alter astrocytic Cl - levels in a number of ways; via (a) the release of neurotransmitters and activation of glial transporters that have intrinsic Cl - conductance, (b) the metabotropic receptor-driven changes in activity of the electroneutral cation-Cl - cotransporter NKCC1, and (c) the transient, activity-dependent changes in glial cell volume which open the volume-regulated Cl - /anion channel VRAC. Reciprocally, astrocytes are thought to alter neuronal [Cl - ] i through either (a) VRAC-mediated release of the inhibitory gliotransmitters, GABA and taurine, which open neuronal GABA A and glycine receptor/Cl - channels, or (b) the gliotransmitter-driven stimulation of NKCC1. The most important recent developments in this area are the identification of the molecular composition and functional heterogeneity of brain VRAC channels, and the discovery of a new cytosolic [Cl - ] sensor - the Wnk family protein kinases. With new work in the field, our understanding of the role of Cl - in information processing within the CNS is expected to be significantly updated. Copyright © 2018 Elsevier B.V. All rights reserved.

Atração e penetração de Meloidogyne javanica e Heterodera glycines em raízes excisadas de soja Attraction and penetration of Meloidogyne javanica and Heterodera glycines in excised soybean roots

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Hercules Diniz Campos

2011-09-01

Full Text Available Com vista ao estudo de atração e penetração de Meloidogyne javanica (Treub Chitwood e Heterodera glycines (Ichinoe em soja (Glycine max L., desenvolveu-se uma técnica empregando-se segmento de raiz com 2cm de comprimento. Nos segmentos de raiz de soja infectados, observou-se que a penetração de juvenis de segundo estádio (J2 de M. javanica ocorre pela coifa seguida de migração entre os feixes vasculares do cilindro central. Juvenis de H. glycines penetraram, aproximadamente, 15mm da coifa. A região seccionada da raiz de soja atraiu três vezes mais J2 de M. javanica do que a região da coifa, mas esta não foi tão atrativa para J2 de H. glycines. A obstrução conjunta da coifa e do local seccionado reduziu (83% a penetração de J2, tanto de M. javanica quanto de H. glycines. Quando apenas um desses locais foi obstruído, a outra extremidade livre compensou o processo atrativo. Portanto, as substâncias atrativas são liberadas por essas extremidades. A penetração de J2 de M. javanica foi maior no segmento de raiz quando comparada com a plântula intacta de soja. Entretanto, os J2 de H. glycines penetraram menos em segmentos de raiz e em plântulas sem folhas, quando comparados com plântulas intactas e com as seccionadas no colo. Portanto, na cultivar de soja "Embrapa 20", a atração e os locais de penetração de J2 de H. glycines e M. javanica são diferenciados. Esta técnica poderá ser útil nos estudos de atração e penetração de outros nematoides endoparasitas.To study the attraction and penetration of Meloidogyne javanica (Treub Chitwood and Heterodera glycines (Ichinoe in soybean (Glycine max L., a technique using 2-cm long root segments was developed. In infected soybean root segments penetration of second stage juveniles (J2 of M. javanica occured through the root cap following migration between the vascular bundles of the central cylinder. Juveniles of H. glycines penetrated about 15mm from the root cap. The cut

Ion Channels and Zinc: Mechanisms of Neurotoxicity and Neurodegeneration

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Deborah R. Morris

2012-01-01

Full Text Available Ionotropic glutamate receptors, such as NMDA, AMPA and kainate receptors, are ligand-gated ion channels that mediate much of the excitatory neurotransmission in the brain. Not only do these receptors bind glutamate, but they are also regulated by and facilitate the postsynaptic uptake of the trace metal zinc. This paper discusses the role of the excitotoxic influx and accumulation of zinc, the mechanisms responsible for its cytotoxicity, and a number of disorders of the central nervous system that have been linked to these neuronal ion channels and zinc toxicity including ischemic brain injury, traumatic brain injury, and epilepsy.

Depression of presynaptic excitation by the activation of vanilloid receptor 1 in the rat spinal dorsal horn revealed by optical imaging

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Ikeda Hiroshi

2006-02-01

Full Text Available Abstract In this study, we show that capsaicin (CAP depresses primary afferent fiber terminal excitability by acting on vanilloid receptor 1 (TRPV1 channels of primary afferent fibers in adenosine 5'-triphosphate (ATP- and temperature-dependent manner using two optical imaging methods. First, transverse slices of spinal cord were stained with a voltage-sensitive dye and the net excitation in the spinal dorsal horn was recorded. Prolonged treatment (>20 min with the TRPV1 channel agonist, CAP, resulted in a long-lasting inhibition of the net excitation evoked by single-pulse stimulation of C fiber-activating strength. A shorter application of CAP inhibited the excitation in a concentration-dependent manner and the inhibition was reversed within several minutes. This inhibition was Ca++-dependent, was antagonized by the TRPV1 channel antagonist, capsazepine (CPZ, and the P2X and P2Y antagonist, suramin, and was facilitated by the P2Y agonist, uridine 5'-triphosphate (UTP. The inhibition of excitation was unaffected by bicuculline and strychnine, antagonists of GABAA and glycine receptors, respectively. Raising the perfusate temperature to 39°C from 27°C inhibited the excitation (-3%/°C. This depressant effect was antagonized by CPZ and suramin, but not by the P2X antagonist, 2', 3'-O-(2,4,6-trinitrophenyl adenosine 5'-triphosphate (TNP-ATP. Second, in order to record the presynaptic excitation exclusively, we stained the primary afferent fibers anterogradely from the dorsal root. CAP application and a temperature increase from 27°C to 33°C depressed the presynaptic excitation, and CPZ antagonized these effects. Thus, this study showed that presynaptic excitability is modulated by CAP, temperature, and ATP under physiological conditions, and explains the reported central actions of CAP. These results may have clinical importance, especially for the control of pain.

Unlike pregnant adult women, pregnant adolescent girls cannot maintain glycine flux during late pregnancy because of decreased synthesis from serine.

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Hsu, Jean W; Thame, Minerva M; Gibson, Raquel; Baker, Tameka M; Tang, Grace J; Chacko, Shaji K; Jackson, Alan A; Jahoor, Farook

2016-03-14

During pregnancy, glycine and serine become more important because they are the primary suppliers of methyl groups for the synthesis of fetal DNA, and more glycine is required for fetal collagen synthesis as pregnancy progresses. In an earlier study, we reported that glycine flux decreased by 39% from the first to the third trimester in pregnant adolescent girls. As serine is a primary precursor for glycine synthesis, the objective of this study was to measure and compare glycine and serine fluxes and inter-conversions in pregnant adolescent girls and adult women in the first and third trimesters. Measurements were made after an overnight fast by continuous intravenous infusions of 2H2-glycine and 15N-serine in eleven adolescent girls (17·4 (se 0·1) years of age) and in ten adult women (25·8 (se 0·5) years of age) for 4 h. Adolescent girls had significantly slower glycine flux and they made less glycine from serine in the third (Padolescent girls (P=0·04) and was significantly associated with third trimester glycine flux. These findings suggest that the pregnant adolescent cannot maintain glycine flux in late pregnancy compared with early pregnancy because of decreased synthesis from serine. It is possible that the inability to maintain glycine synthesis makes her fetus vulnerable to impaired cartilage synthesis, and thus linear growth.

Negative modulation of NMDA receptor channel function by DREAM/calsenilin/KChIP3 provides neuroprotection?

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Wang, KeWei; Wang, Yun

2012-01-01

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels highly permeable to calcium and essential to excitatory neurotransmission. The NMDARs have attracted much attention because of their role in synaptic plasticity and excitotoxicity. Evidence has recently accumulated that NMDARs are negatively regulated by intracellular calcium binding proteins. The calcium-dependent suppression of NMDAR function serves as a feedback mechanism capable of regulating subsequent Ca2+ entry into the postsynaptic cell, and may offer an alternative approach to treating NMDAR-mediated excitotoxic injury. This short review summarizes the recent progress made in understanding the negative modulation of NMDAR function by DREAM/calsenilin/KChIP3, a neuronal calcium sensor (NCS) protein. PMID:22518099

15N-labelled glycine synthesis

International Nuclear Information System (INIS)

Tavares, Claudineia R.O.; Bendassolli, Jose A.; Sant'Ana Filho, Carlos R.; Prestes, Clelber V.; Coelho, Fernando

2006-01-01

This work describes a method for 15 N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of α-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia ( 15 NH 3 ). Special care was taken to avoid possible 15 NH 3 losses, since its production cost is high. In that respect, although the purchase cost of the 13 N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an important tool for N cycling studies in living organisms, also minimizing labor and environmental hazards, as well as time limitation problems in field studies. The tests were carried out with three replications, and variable 15 NH 3(aq) volumes in the reaction were used (50, 100, and 150 mL), in order to calibrate the best operational condition; glycine masses obtained were 1.7, 2, and 3.2 g, respectively. With the development of a system for 15 NH 3 recovery, it was possible to recover 71, 83, and 87% of the ammonia initially used in the synthesis. With the required adaptations, the same system was used to recover methanol, and 75% of the methanol initially used in the amino acid purification process were recovered. (author)

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

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Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

2015-12-01

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

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Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Korn, Matthew J; Nakamura, Paul A; Shirkey, Nicole J; Wong, Jamie K; Miledi, Ricardo

2013-03-22

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization.

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Nicole K McKinnon

Full Text Available 5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4 and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-A(n = 2-7. Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC(50's were at most 5-fold greater than wild-type (WT. The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A(2, 5-HT3A-A(4, 5-HT3A-A(6, and 5-HT3A-A(7 were similar to WT. In contrast, 5-HT3A-A(3 and 5-HT3A-A(5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A(1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

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Varju, P; Schlett, K; Eisel, U; Madarász, E

2001-06-01

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Ion channels: molecular targets of neuroactive insecticides.

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Raymond-Delpech, Valérie; Matsuda, Kazuhiko; Sattelle, Benedict M; Rauh, James J; Sattelle, David B

2005-11-01

Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p'-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); gamma-aminobutyric acid (GABA) receptors (cyclodienes, gamma-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.

Electrodeposition of CoNiMo thin films using glycine as additive: anomalous and induced codeposition

International Nuclear Information System (INIS)

Esteves, Marcos C.; Sumodjo, Paulo T.A.; Podlaha, Elizabeth J.

2011-01-01

Highlights: → Mixed/induced codeposition of CoNiMo from a glycine containing bath. → Deposition in a rotating cylinder Hull cell. → The mechanism is explained in term of the complex species that can be formed. - Abstract: The present study focuses on the behavior of the CoNiMo mixed anomalous/induced codeposition process, using glycine as a probe to influence the coverage of adsorbed intermediates. In order to facilitate the investigation of a wide variation of parameters the electrodeposition of the alloy films was performed using a rotating cylinder Hull cell. Alloy composition, current efficiency and partial currents of each metal were analyzed. The partial current densities and hence alloy composition was affected by the amount of glycine in the electrolyte: increasing glycine enhanced both cobalt and molybdenum deposition rates and hindered nickel deposition. It is suggested that the glycine facilitates the adsorption of M(I) adsorbed intermediates that control the anomalous and induced codeposition behavior. The current efficiency ranged from 30 up to 75% and was only slightly affected by glycine at high applied current densities. Films with a tridimensional porous structure were obtained applying current densities higher than 200 mA cm -2 , formed as a consequence of the large hydrogen evolution side reaction, presenting conditions for a novel Mo-alloy electrode structure.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity

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Douglas Bledsoe

2017-05-01

Full Text Available N-methyl D-aspartate receptors (NMDAR play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D and two GluN3 (A–B subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD and ligand binding domain (LBD, transmembrane domain (TMD and an intracellular C-terminal domain (CTD. Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

A conserved aspartic acid is important for agonist (VUAA1 and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco.

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Brijesh N Kumar

Full Text Available Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco. An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca(2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ~2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.

Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine

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Liao, Yi-Ting; Manson, Anthony C.; DeLyser, Michael R.; Noid, William G.; Cremer, Paul S.

2017-01-01

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air–water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer–water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins. PMID:28228526

Glycine Betaine Recognition through Cation−π Interactions in Crystal Structures of Glycine Betaine Complexes with C-Ethyl-pyrogallol[4]arene and C-Ethyl-resorcin[4]arene as Receptors

Directory of Open Access Journals (Sweden)

Ikuhide Fujisawa

2013-04-01

Full Text Available The glycine betaine (betaine, interacts with several types of proteins with diverse structures in vivo, and in the contact regions, the aromatic rings of protein residues are frequently found beside the trimethylammonium group of betaine, implying the importance of the cation−π interactions in recognition of this molecule. The crystal structures determined by X-ray crystallography of the complexes of betaine and C-ethyl-pyrogallol[4]arene (pyrogallol cyclic tetramer: PCT and betaine and C-ethyl-resorcin[4]arene (resorcinol cyclic tetramer: RCT mimic the conformations of betaine and protein complexes and show that the clathrate conformations are retained by the cation−π interactions. The difference of the conformation feature of betaine in the Protein Data Bank and in the Cambridge Structural Database was found by chance during the research and analyzed with the torsion angles.

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate

Czech Academy of Sciences Publication Activity Database

Ladislav, Marek; Černý, Jiří; Krůšek, Jan; Balík, Aleš; Vyklický ml., Ladislav

2018-01-01

Roč. 11, Apr 6 (2018), č. článku 113. ISSN 1662-5099 R&D Projects: GA ČR(CZ) GA17-02300S; GA TA ČR(CZ) TE01020028; GA MZd(CZ) NV15-29370A; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : glutamate receptor gating * electrophysiology * spontaneous activity * channel open probability * protein block alphabet * molecular modeling Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 5.076, year: 2016

High efficient removal of chromium (VI) using glycine doped polypyrrole adsorbent from aqueous solution

CSIR Research Space (South Africa)

Ballav, N

2012-08-01

Full Text Available Glycine doped polypyrrole (PPy-gly) adsorbent was prepared via in situ polymerization of pyrrole (Py) monomer in the presence of glycine (gly) for the removal of Cr(VI). Formation of PPy homopolymer and inclusion of gly in the PPy matrix were...

Application of Glycine-TTC dosimeter in gamma radiation processing facility

International Nuclear Information System (INIS)

Shinde, S.H.; Mondal, S.; Kulkarni, M.S.

2018-01-01

Glycine-TTC dosimeter was found to have a useful dose range of 5 to 30 kGy using spectro-photometric read-out method. Potential use of this dosimeter was demonstrated by measuring dose-rate in gamma chamber GC 900. The aim of the present study was to verify the performance of this dosimeter in actual industrial processing conditions encountered in radiation processing facility such as Gamma Radiation Processing Plant for Spices (GRPPS), BRIT, Vashi. Accordingly, glycine-TTC dosimeters were irradiated along with routine dosimeter viz. ceric-cerous of GRPPS and reference standard dosimeter viz. alanine EPR

New insights into the catalytic mechanism of human glycine N-acyltransferase.

Science.gov (United States)

van der Sluis, Rencia; Ungerer, Vida; Nortje, Carla; A van Dijk, Alberdina; Erasmus, Elardus

2017-11-01

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism. © 2017 Wiley Periodicals, Inc.

ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

Science.gov (United States)

Giffin, Michelle M.; Modesti, Lucia; Raab, Ronald W.; Wayne, Lawrence G.

2012-01-01

The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown. PMID:22210765

Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

Science.gov (United States)

Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

1991-06-01

RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

Isolation, Characterization, and Distribution of a Biocontrol Fungus from Cysts of Heterodera glycines.

Science.gov (United States)

Kim, D G; Riggs, R D; Correll, J C

1998-05-01

ABSTRACT Seventy-six populations of Heterodera glycines were collected from 33 counties in 10 states of the United States along the Mississippi and Missouri Rivers in 1992 and 1993. A sterile hyphomycete fungus of an unnamed taxon, designated ARF18 and shown to be a parasite of eggs of H. glycines, was isolated from eggs and cysts of 10 of the populations from Kentucky, Louisiana, Mississippi, and Tennessee. Ten isolates of ARF18 obtained in this study and seven isolates obtained in earlier studies were characterized for cultural morphology on several growth media, the ability to produce sclerotium-like structures (SLS) on cornmeal agar, growth rates, pathogenicity to eggs of H. glycines in vitro, and mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs). All 17 isolates of ARF18 readily grew on potato dextrose agar, cornmeal agar, and nutrient agar. Based on colony morphology and SLS appearance on cornmeal agar, the isolates could be grouped into two morphological phenotypes. Isolates that produced SLS that were composed of a compact mass of hyphae were designated ARF18-C, whereas isolates that produced SLS composed of a mass of loosely clumped hyphae were designated ARF18-L. Only minor differences in growth rates were detected among the ARF18-C and ARF18-L isolates. All 10 ARF18-C isolates, which were from Arkansas, Louisiana, Mississippi, and Tennessee, belonged to a single mtDNA RFLP haplotype. The seven ARF18-L isolates shared many comigrating mtDNA restriction fragments with one another, but belonged to three distinct mtDNA RFLP haplotypes. Ability to infect eggs of H. glycines in vitro varied considerably among the various isolates of ARF18. In particular, several of the ARF18-C isolates were consistently able to infect over 50% (mean = 70.0%, standard deviation = 16%) of the eggs of H. glycines, whereas ARF18-L infected eggs to a lesser degree (mean = 25%, standard deviation = 27%). ARF18-C was isolated only from H. glycines populations

Mapping allostery through computational glycine scanning and correlation analysis of residue-residue contacts.

Science.gov (United States)

Johnson, Quentin R; Lindsay, Richard J; Nellas, Ricky B; Fernandez, Elias J; Shen, Tongye

2015-02-24

Understanding allosteric mechanisms is essential for the physical control of molecular switches and downstream cellular responses. However, it is difficult to decode essential allosteric motions in a high-throughput scheme. A general two-pronged approach to performing automatic data reduction of simulation trajectories is presented here. The first step involves coarse-graining and identifying the most dynamic residue-residue contacts. The second step is performing principal component analysis of these contacts and extracting the large-scale collective motions expressed via these residue-residue contacts. We demonstrated the method using a protein complex of nuclear receptors. Using atomistic modeling and simulation, we examined the protein complex and a set of 18 glycine point mutations of residues that constitute the binding pocket of the ligand effector. The important motions that are responsible for the allostery are reported. In contrast to conventional induced-fit and lock-and-key binding mechanisms, a novel "frustrated-fit" binding mechanism of RXR for allosteric control was revealed.

Dental enamel cells express functional SOCE channels.

Science.gov (United States)

Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

2015-10-30

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity

DEFF Research Database (Denmark)

Lund, Trine Meldgaard; Ploug, K.B.; Iversen, Anne

2015-01-01

-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown...... an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β...... to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release....

An evolutionarily conserved gene family encodes proton-selective ion channels.

Science.gov (United States)

Tu, Yu-Hsiang; Cooper, Alexander J; Teng, Bochuan; Chang, Rui B; Artiga, Daniel J; Turner, Heather N; Mulhall, Eric M; Ye, Wenlei; Smith, Andrew D; Liman, Emily R

2018-03-02

Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3 , and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The discovery of glycine and related amino acid-based factor Xa inhibitors

Energy Technology Data Exchange (ETDEWEB)

Kohrt, Jeffrey T.; Filipski, Kevin J.; Cody, Wayne L.; Bigge, Christopher F.; La, Frances; Welch, Kathleen; Dahring, Tawny; Bryant, John W.; Leonard, Daniele; Bolton, Gary; Narasimhan, Lakshmi; Zhang, Erli; Peterson, J. Thomas; Haarer, Staci; Sahasrabudhe, Vaishali; Janiczek, Nancy; Desiraju, Shrilakshmi; Hena, Mostofa; Fiakpui, Charles; Saraswat, Neerja; Sharma, Raman; Sun, Shaoyi; Maiti, Samarendra N.; Leadley, Robert; Edmunds, Jeremy J. (Naeja); (Pfizer)

2010-12-03

Herein, we report on the identification of three potent glycine and related amino acid-based series of FXa inhibitors containing a neutral P1 chlorophenyl pharmacophore. A X-ray crystal structure has shown that constrained glycine derivatives with optimized N-substitution can greatly increase hydrophobic interactions in the FXa active site. Also, the substitution of a pyridone ring for a phenylsulfone ring in the P4 sidechain resulted in an inhibitor with enhanced oral bioavailability.

Transient receptor potential channel ankyrin-1 is not a cold sensor for autonomic thermoregulation in rodents.

Science.gov (United States)

de Oliveira, Cristiane; Garami, Andras; Lehto, Sonya G; Pakai, Eszter; Tekus, Valeria; Pohoczky, Krisztina; Youngblood, Beth D; Wang, Weiya; Kort, Michael E; Kym, Philip R; Pinter, Erika; Gavva, Narender R; Romanovsky, Andrej A

2014-03-26

The rodent transient receptor potential ankyrin-1 (TRPA1) channel has been hypothesized to serve as a temperature sensor for thermoregulation in the cold. We tested this hypothesis by using deletion of the Trpa1 gene in mice and pharmacological blockade of the TRPA1 channel in rats. In both Trpa1(-/-) and Trpa1(+/+) mice, severe cold exposure (8°C) resulted in decreases of skin and deep body temperatures to ∼8°C and 13°C, respectively, both temperatures being below the reported 17°C threshold temperature for TRPA1 activation. Under these conditions, Trpa1(-/-) mice had the same dynamics of body temperature as Trpa1(+/+) mice and showed no weakness in the tail skin vasoconstriction response or thermogenic response to cold. In rats, the effects of pharmacological blockade were studied by using two chemically unrelated TRPA1 antagonists: the highly potent and selective compound A967079, which had been characterized earlier, and the relatively new compound 43 ((4R)-1,2,3,4-tetrahydro-4-[3-(3-methoxypropoxy)phenyl]-2-thioxo-5H-indeno[1,2-d]pyrimidin-5-one), which we further characterized in the present study and found to be highly potent (IC50 against cold of ∼8 nm) and selective. Intragastric administration of either antagonist at 30 mg/kg before severe (3°C) cold exposure did not affect the thermoregulatory responses (deep body and tail skin temperatures) of rats, even though plasma concentrations of both antagonists well exceeded their IC50 value at the end of the experiment. In the same experimental setup, blocking the melastatin-8 (TRPM8) channel with AMG2850 (30 mg/kg) attenuated cold-defense mechanisms and led to hypothermia. We conclude that TRPA1 channels do not drive autonomic thermoregulatory responses to cold in rodents.

Kinetics and mechanism of oxidation of glycine by iron(III)–1,10 ...

Indian Academy of Sciences (India)

Unknown

An increase in (phenanthroline) increases the rate, while increase in [H+] decreases the rate. ... bon dioxide and ammonia with K2S2O8, KMnO4, po- ... 2. Experimental. A 1⋅0 mol dm–3 solution of glycine is prepared afresh by dissolving glycine (E-Merck) in water and its strength is determined by the acetuous perchloric.

Synthesis and evaluation of 1-[l brace]1-[5-(2'-[[sup 18]F]fluoroethyl)-2-thienyl]cyclohexyl[r brace]piperidine as a potential in vivo radioligand for the NMDA receptor-channel complex. [N-methyl-D-aspartate receptor

Energy Technology Data Exchange (ETDEWEB)

Orita, Kazuhiro; Sasaki, Shigeki; Maeda, Minoru [Kyushu Univ., Fukuoka (Japan). Faculty of Pharmaceutical Sciences; Hashimoto, Atsushi; Nishikawa, Toru [National Inst. of Neuroscience, Tokyo (Japan); Yugami, Tomoko; Umezu, Kohei [Mitsubishi Kasei Corp., Yokohama (Japan). Research Center

1993-10-01

1-[l brace]1-[5-(2'-[[sup 18]F]Fluoroethyl)-2-thienyl]cyclohexyl[r brace]piperidine ([sup 18]FE-TCP) was prepared a fluorine-substituted analogue of the potent NMDA receptor channel blocker, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), by the mesylate displacement with [[sup 18]F]fluoride ion with isolated radiochemical yields of 6-12%, and the synthesis time including a two step HPLC purification was 120 min. The regional distribution in rat brain after i.v. injection of [sup 18]FE-TCP was heterogeneous and similar to the known distribution of phencyclidine recognition sites, with hippocampus-cerebellum, striatum-cerebellum and cerebral cortex-cerebellum concentration ratios of 2.08, 1.7 and 1.54, respectively, 15 min post-injection. Furthermore, this localized regional cerebral distribution was blocked by co-injection with the unlabelled FE-TCP or pretreatment with cis-2-hydroxymethyl-r-1-(N-piperidyl)-1-(2-thienyl)cyclohexane, with the greatest reductions seen in the hippocampus followed by the striatum and cerebral cortex. However, relatively low receptor binding affinity and high non-specific binding due to its high lipophilicity suggest that [sup 18]FE-TCP may not be a suitable radioligand for in vivo PET investigations of the NMDA receptor-channel complex. (Author).

RNA-Sequencing Analyses Demonstrate the Involvement of Canonical Transient Receptor Potential Channels in Rat Tooth Germ Development

Directory of Open Access Journals (Sweden)

Jun Yang

2017-06-01

Full Text Available Tooth development depends on multiple molecular interactions between the dental epithelium and mesenchyme, which are derived from ectodermal and ectomesenchymal cells, respectively. We report on a systematic RNA sequencing analysis of transcriptional expression levels from the bud to hard tissue formation stages of rat tooth germ development. We found that GNAO1, ENO1, EFNB1, CALM1, SIAH2, ATP6V0A1, KDELR2, GTPBP1, POLR2C, SORT1, and members of the canonical transient receptor potential (TRPC channel family are involved in tooth germ development. Furthermore, Cell Counting Kit 8 (CCK8 and Transwell migration assays were performed to explore the effects of these differentially expressed genes (DEGs on the proliferation and migration of dental pulp stem cells. Immunostaining revealed that TRPC channels are expressed at varying levels during odontogenesis. The identified genes represent novel candidates that are likely to be vital for rat tooth germ development. Together, the results provide a valuable resource to elucidate the gene regulatory mechanisms underlying mammalian tooth germ development.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

Science.gov (United States)

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Resveratrol, tryptophanum, glycine and vitamin E: a nutraceutical approach to sleep disturbance and irritability in peri- and post-menopause.

Science.gov (United States)

Parazzini, F

2015-02-01

The climacteric syndrome is characterized by several symptoms: hot flashes are the most common and reported by about 70% of peri- post-menopausal women. Sleep disorders, particularly decreased sleep quality, and irritability are also commonly reported. There is a clinical and epidemiological relationship between these symptoms. Common biological mechanisms may explain in part the relationship between hot flushes, sleep disorders and irritability. For example, withdrawal of hormones causes change in the serotonin levels. Tryptophan is an essential amino acid. it is the precursor for the serotonin synthesis and is naturally found in food such as turkey, cheese, and nuts. The serotonergic system is implicated in sleep, mood, and hot flashes. Glycine is an amino acid found mainly in protein-rich food such as meat, fish, dairy products, cheese and vegetables. It is an inhibitory neurotransmitter in the central nervous system. Studies have shown that glycine can promote a deeper level of sleep. Resveratrol has a similar chemical structure to the diethylstilbestrol and 17-beta estradiol and acts as a phytoestrogen. Resveratrol at doses of 3-10 micromoles inhibited the estradiol-estrogen receptor binding and showed an estrogen-like activity. Vitamin E is found naturally in some food and available as a dietary supplement. It has an antioxidant activity. It has been suggested that the oxidative stress may also play a role in sleep disorders. Some studies have shown protective effect of vitamins E on sleep quality. In conclusion, hot flashes, sleep disturbances and mood disorders may represent a continuum in the climacteric syndrome, which recognize in the hormonal changes and the neurotrasmettitors level alteration a potential common pathway. The nutraceutical approach may be useful in a preventive perspective. Among the large choice of functional food available, the combination of resveratrol, tryptophanum, glycine and vitamin E may represent an interesting opportunity in

Different effects of pentobarbital on two γ-aminobutyrate receptors from rat brain: channel opening, desensitization, and an additional conformational change