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Sample records for voltage-dependent sodium channel

  1. A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms.

    Science.gov (United States)

    Hofer, Doris; Lohberger, Birgit; Steinecker, Bibiane; Schmidt, Kurt; Quasthoff, Stefan; Schreibmayer, Wolfgang

    2006-05-24

    The specific, acute interaction of tolperisone, an agent used as a muscle relaxant and for the treatment of chronic pain conditions, with the Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8) isoforms of voltage dependent sodium channels was investigated and compared to that of lidocaine. Voltage dependent sodium channels were expressed in the Xenopus laevis oocyte expression system and sodium currents were recorded with the two electrode voltage clamp technique. Cumulative dose response relations revealed marked differences in IC(50) values between the two drugs on identical isoforms, as well as between isoforms. A detailed kinetic analysis uncovered that tolperisone as well as lidocaine exhibited their blocking action not only via state dependent association/dissociation with voltage dependent sodium channels, but a considerable fraction of inhibition is tonic, i.e. permanent and basic in nature. Voltage dependent activation was affected to a minor extent only. A shift in steady-state inactivation to more negative potentials could be observed for most drug/isoform combinations. The contribution of this shift to overall block was, however, small at drug concentrations resulting in considerable overall block. Recovery from inactivation was affected notably by both drugs. Lidocaine application led to a pronounced prolongation of the time constant of the fast recovery process for the Na(v1.3), Na(v1.5), and Na(v1.7) isoforms, indicating common structural properties in the local anesthetic receptor site of these three proteins. Interestingly, this characteristic drug action was not observed for tolperisone.

  2. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    Energy Technology Data Exchange (ETDEWEB)

    Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Clare, Jeffrey J. [Eaton Pharma Consulting, Eaton Socon, Cambridgeshire PE19 8EF (United Kingdom); Debanne, Dominique [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Alcaraz, Gisele, E-mail: gisele.alcaraz@univmed.fr [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France)

    2011-07-29

    Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.

  3. The Mechanism of Voltage Dependent Gating of the NaChBac Prokaryotic Sodium Channel

    Science.gov (United States)

    Decaen, Paul G.

    Electrical signaling in cells depends on selective conductance of ions through membrane proteins called 'voltage gated ion channels'. These channels are characterized by their ability turn on and off the flow of ionic current by opening and closing their conductive pore in response to changes in membrane potential. The opening and closing of the pore is a mechanically linked to conformational movement of the positively charged fourth transmembrane segment (S4) in 'the voltage sensor' region. How the S4 moves in response to membrane potential is a controversial subject. In this thesis, we used the prokaryotic sodium channel NaChBac as our model sodium channel to study voltage dependent movement of the S4 in the voltage sensor. We use a disulfide-locking method where we introduced pairs of cysteines in the voltage sensor that crosslink and trap the S4 in its path after depolarization. We screened over one hundred mutations of the NaChBac channel in the whole cell patch clamp assay and demonstrated discrete and sequential voltage dependent ion pair interactions that occur in at least three states between the positively charged residues of the S4 segment and the acidic residues in the S1, S2 and S3 segments. In conjunction with structural modeling of the voltage sensor and our disulfide locking data, we propose that the S4 moves in and out of the plane of the membrane 8-13 A, forming distinct gating charge interactions with counter charges of the voltage sensor and adopts a 310 helix over a portion of its structure during activation. These findings are compatible with the sliding helix model and refine our understanding of the structural determinates of voltage sensor function in voltage gated ion channels.

  4. Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

    Science.gov (United States)

    Angelides, K J; Nutter, T J; Elmer, L W; Kempner, E S

    1985-03-25

    Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

  5. Functional coupling between sodium-activated potassium channels and voltage-dependent persistent sodium currents in cricket Kenyon cells.

    Science.gov (United States)

    Takahashi, Izumi; Yoshino, Masami

    2015-10-01

    In this study, we examined the functional coupling between Na(+)-activated potassium (KNa) channels and Na(+) influx through voltage-dependent Na(+) channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Single-channel activity of KNa channels was recorded with the cell-attached patch configuration. The open probability (Po) of KNa channels increased with increasing Na(+) concentration in a bath solution, whereas it decreased by the substitution of Na(+) with an equimolar concentration of Li(+). The Po of KNa channels was also found to be reduced by bath application of a high concentration of TTX (1 μM) and riluzole (100 μM), which inhibits both fast (INaf) and persistent (INaP) Na(+) currents, whereas it was unaffected by a low concentration of TTX (10 nM), which selectively blocks INaf. Bath application of Cd(2+) at a low concentration (50 μM), as an inhibitor of INaP, also decreased the Po of KNa channels. Conversely, bath application of the inorganic Ca(2+)-channel blockers Co(2+) and Ni(2+) at high concentrations (500 μM) had little effect on the Po of KNa channels, although Cd(2+) (500 μM) reduced the Po of KNa channels. Perforated whole cell clamp analysis further indicated the presence of sustained outward currents for which amplitude was dependent on the amount of Na(+) influx. Taken together, these results indicate that KNa channels could be activated by Na(+) influx passing through voltage-dependent persistent Na(+) channels. The functional significance of this coupling mechanism was discussed in relation to the membrane excitability of Kenyon cells and its possible role in the formation of long-term memory.

  6. Neuroprotective activity of stiripentol with a possible involvement of voltage-dependent calcium and sodium channels.

    Science.gov (United States)

    Verleye, Marc; Buttigieg, Dorothée; Steinschneider, Rémy

    2016-02-01

    A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage-gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ-aminobutyric acidergic properties, on neuronal-astroglial cultures from rat cerebral cortex exposed to oxygen-glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10-100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC50  >100 µM) but significantly blocked the entry of Na(+) and Ca(2+) activated by veratridine and NMDA, respectively. These results suggest that Na(+) and Ca(2+) channels could contribute to the neuroprotective properties of sitiripentol.

  7. Inhibition of rat hippocampal excitability by the plant alkaloid 3-acetylaconitine mediated by interaction with voltage-dependent sodium channels.

    Science.gov (United States)

    Ameri, A

    1997-02-01

    The effects of the Aconitum alkaloid 3-acetylaconitine on neuronal activity were investigated in the slice preparation and on cultivated neurons of rat hippocampus by extracellular and patch-clamp recordings, respectively. 3-Acetylaconitine (0.01-1 microM) diminished the orthodromic and antidromic population spike in a concentration-dependent manner. The inhibitory action of the drug was preceded by a transiently enhanced excitability. The latency of onset of the inhibition was accelerated by increased stimulation frequency, whereas recovery during washout of the alkaloid was accelerated by decreased stimulation frequency. Moreover, the inhibitory effect of 3-acetylaconitine was evaluated in two different models of epileptiform activity induced either by blockade of GABA receptors by bicuculline (10 microM) or by a nominal Mg(2+)-free bathing medium. In accordance with the activity-dependent mode of action, this compound abolished the synaptically evoked population spikes in the presence of bicuculline or nominal Mg(2+)-free bathing medium, respectively. Whole-cell patch-clamp recordings revealed an interaction of 3-acetylaconitine with the voltage-dependent sodium channel. At a concentration of 1 microM, 3-acetylaconitine did not affect the peak amplitude of the sodium current, but shifted the current-voltage relationship in the hyperpolarized direction such that sodium currents were already activated at the resting potential.

  8. Cysteine mutagenesis in the voltage-dependent sodium channel structural insights and implications.

    Science.gov (United States)

    Tomaselli, G F

    1997-08-01

    The superfamily of ion channel proteins comprise multisubunit transmembrane glycoproteins that are the fundamental electrical signaling molecules in the heart and other excitable tissues. The large size and hydrophobicity of these proteins present a formidable obstacle to the generation of a crystal structure. In lieu of a high-resolution structure, complementary methods have been used to study the structure function relationships of these essential excitability proteins. Molecular cloning and biophysical analysis of heterologously expressed wild-type and mutant channel proteins have provided insights into the structural basis of the essential channel functions of permeation and gating. This powerful combination of techniques also provides dynamic structural information regarding channel proteins not likely to be forthcoming from a crystal structure. (Trends Cardiovasc Med 1997;7:211-218). © 1997, Elsevier Science Inc.

  9. Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels.

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    Castañeda-Castellanos, David R; Nikonorov, Igor; Kallen, Roland G; Recio-Pinto, E

    2002-03-28

    We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.

  10. Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery.

    Science.gov (United States)

    Rusinova, Radda; Herold, Karl F; Sanford, R Lea; Greathouse, Denise V; Hemmings, Hugh C; Andersen, Olaf S

    2011-08-01

    The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator-activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer-water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs' partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer-dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and

  11. Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors.

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    Darbon, H; Angelides, K J

    1984-05-25

    A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.

  12. Quantum entanglement in the voltage dependent sodium channel can reproduce the salient features of neuronal action potential initiation

    CERN Document Server

    Summhammer, Johann

    2007-01-01

    We investigate the effects of a quantum entanglement regime within an ion conducting molecule (ion channel) of the neuronal plasma membrane on the onset dynamics of propagating nerve pulses (action potentials). In particular, we model the onset parameters of the sodium current in the Hodgkin Huxley equation as three similar but independent probabilistic mechanisms which become quantum entangled. The underlying physics is general and can involve entanglement between various degrees of freedom underlaying ion transition states or 'gating states' during conduction, e.g. Na$^+$ ions in different channel locations, or different 'affinity' states of ions with atoms lining the sub-regions of the channel protein ('filter-states'). We find that the 'quantum corrected' Hodgkin Huxley equation incorporating entangled systems states can reproduce action potential pulses with the critical onset dynamics observed recently in neocortical neurons in vivo by Naundorf et al. [Nature {\\bf 440}, 1060 (20 April 2006)]. Interestin...

  13. Beta-scorpion toxin effects suggest electrostatic interactions in domain II of voltage-dependent sodium channels. : Electrostatic interactions between segments IIS2, IIS3 and IIS4 of Na+ channel.

    OpenAIRE

    Mantegazza, Massimo; Cestèle, Sandrine

    2005-01-01

    International audience; Beta-scorpion toxins specifically modulate the voltage dependence of sodium channel activation by acting through a voltage-sensor trapping model. We used mutagenesis, functional analysis and the action of beta-toxin as tools to investigate the existence and role in channel activation of molecular interactions between the charged residues of the S2, S3 and S4 segments in domain II of sodium channels. Mutating to arginine the acidic residues of the S2 and S3 transmembran...

  14. [Role of voltage-dependent ion channels in epileptogenesis].

    Science.gov (United States)

    Ricard-Mousnier, B; Couraud, F

    1993-10-01

    The aim of this review is to gather information in favour of the involvement of voltage-dependent ion channels in epileptogenesis. Although, up to now, no study has shown that epilepsy is accompanied by a modification in the activity to these channels, the recently acquired knowledge of their physiology allows to presume would favor their involvement in epileptogenesis. The results from electrophysiological studies are as follows: a persistent sodium current increases neuronal excitability whereas potassium currents have an inhibitory role. In particular, calcium-dependent potassium current are involved in the post-hyperpolarization phases which follows PDS. Calcium currents are also involved in the genesis of the "bursting pacemaker" activity displayed by the neurons presumed to be inducers of the epileptic activity. Biochemical data has shown that as a consequence of epileptic activity, sodium and calcium channels are down regulated. This down-regulation could be a way to reduces neuronal hyperexcitability. Pharmacological data demonstrate the drugs which activate calcium channels or which inhibit potassium channels have a convusilvant effect. On the contrary, agents which block calcium or sodium channels or which properties. Among the latter ones, some antiepileptic drugs can be found. In summary situations which lead to increase in calcium and sodium currents and/or to an inhibition in potassium currents are potentially epileptogenic.

  15. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, R.D.; Fieles, W.E.; Schotland, D.L.; Hogue-Angeletti, R.; Barchi, R.L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

  16. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

    Science.gov (United States)

    Gordon, R D; Fieles, W E; Schotland, D L; Hogue-Angeletti, R; Barchi, R L

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% "right-side-out" as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure. Images PMID:2432607

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  18. Voltage-dependent gating of hERG potassium channels

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    Yen May eCheng

    2012-05-01

    Full Text Available The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4-S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-a-go-go related gene, hERG, which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure-function relationships underlying voltage-dependent gating in Shaker and hERG channels, with a focus on the roles of the voltage sensing domain and the S4-S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter charge interactions. More recent data suggest that key amino acid differences in the hERG voltage sensing unit and S4-S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  20. Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes.

    Science.gov (United States)

    Rubi, Lena; Gawali, Vaibhavkumar S; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz; Koenig, Xaver

    2015-01-01

    Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B). Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf) mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types. © 2015 S. Karger AG, Basel.

  1. Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes

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    Lena Rubi

    2015-06-01

    Full Text Available Background/Aims: Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B. Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. Methods and Results: By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Conclusion: Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types.

  2. Conductance hysteresis in the voltage-dependent anion channel.

    Science.gov (United States)

    Rappaport, Shay M; Teijido, Oscar; Hoogerheide, David P; Rostovtseva, Tatiana K; Berezhkovskii, Alexander M; Bezrukov, Sergey M

    2015-09-01

    Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.

  3. Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Voltage-Dependent Anion Channel (VDAC phosphorylated by c-Jun N-terminal Kinase-3 (JNK3 was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

  4. G Protein-induced Trafficking of Voltage-dependent Calcium Channels

    National Research Council Canada - National Science Library

    Eugene Tombler; Nory Jun Cabanilla; Paul Carman; Natasha Permaul; John J. Hall; Ryan W. Richman; Jessica Lee; Jennifer Rodriguez; Dan P. Felsenfeld; Robert F. Hennigan; María A. Diversé-Pierluissi

    2006-01-01

    .... Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane...

  5. The action of a phorbol ester on voltage-dependent parameters of the sodium current in isolated hippocampal neurons.

    Science.gov (United States)

    Chizhmakov, I V; Klee, M R

    1994-03-01

    The action of a phorbol ester (phorbol-12,13-diacetate) on the voltage-activated sodium current has been investigated by the voltage-clamp method in acutely isolated pyramidal neurons from rat hippocampus. The intracellular perfusion of isolated pyramidal neurons for 30-40 min induced a gradual 10-15 mV shift in both the current-voltage relationship and voltage-dependent steady-state inactivation to more negative potentials. The application of phorbol ester (1-10 microM) to isolated neurons for the same time increased the amplitude of sodium current by 15-20%, shifted the above-mentioned voltage-dependent parameters for an additional 10-15 mV in the same direction and changed the slope of the steady-state inactivation curve. In contrast, after prolonged incubation of slices in the phorbol ester-containing solution (1-10 microM) for 0.5-3 h, subsequent application of phorbol ester at the same concentration caused neither the addition shift of the voltage-dependent characteristics of sodium channels nor the change of the slope of the steady-state inactivation curve. However, in this case an increase in the amplitude of sodium current by 15-20% during 30-40 min intracellular perfusion was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Novel expression and regulation of voltage-dependent potassium channels in placentas from women with preeclampsia.

    Science.gov (United States)

    Mistry, Hiten D; McCallum, Laura A; Kurlak, Lesia O; Greenwood, Iain A; Broughton Pipkin, Fiona; Tribe, Rachel M

    2011-09-01

    Preeclampsia is associated with structural/functional alterations in placental and maternal vasculature. Voltage-dependant potassium channels encoded by KCNQ1-5 genes have been detected in several types of blood vessels where they promote vascular relaxation. Voltage-dependant potassium channel function can be modulated by KCNE1-5-encoded accessory proteins. The aim of this study was to determine whether KCNQ and KCNE genes are differentially expressed in placentas from women with preeclampsia compared with normotensive controls and to examine any differences in those who delivered preterm (voltage-dependant potassium channels are expressed and markedly modulated in placentas from preeclamptic women. Differential expression of isoforms may lead to altered cell proliferation. The correlation between KCNQ3 and KCNE5 expression is indicative of a novel channel complex and warrants further investigation.

  7. Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons.

    Science.gov (United States)

    O'Dowd, D K; Ribera, A B; Spitzer, N C

    1988-03-01

    Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.

  8. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, T M; Belhage, B

    2001-01-01

    using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears...

  9. Voltage-dependent calcium channels from brain incorporated into planar lipid bilayers

    Science.gov (United States)

    Nelson, Mark T.; French, Robert J.; Krueger, Bruce K.

    1984-03-01

    Many important physiological processes, including neurotransmitter release and muscle contraction1-3, are regulated by the concentration of Ca2+ ions in the cell. Levels of cytoplasmic Ca2+ can be elevated by the entry of Ca2+ ions through voltage-dependent channels which are selective for Ca2+, Ba2+ and Sr2+ ions4-14. We have measured currents through single, voltage-dependent calcium channels from rat brain that have been incorporated into planar lipid bilayers. Channel gating was voltage-dependent: membrane depolarization increased the channel open times and decreased the closed times. The channels were selective for divalent cations over monovalent ions. The well-known calcium channel blockers, lanthanum and cadmium, produced a concentration-dependent reduction of the apparent single-channel conductance. Contrary to expectations14, the nature of the divalent cation carrying current through the channel affected not only the single-channel conductance, but also the channel open times, with mean open times being shortest for barium.

  10. Endocytic regulation of voltage-dependent potassium channels in the heart.

    Science.gov (United States)

    Ishii, Kuniaki; Norota, Ikuo; Obara, Yutaro

    2012-01-01

    Understanding the regulation of cardiac ion channels is critical for the prevention of arrhythmia caused by abnormal excitability. Ion channels can be regulated by a change in function (qualitative) and a change in number (quantitative). Functional changes have been extensively investigated for many ion channels including cardiac voltage-dependent potassium channels. By contrast, the regulation of ion channel numbers has not been widely examined, particularly with respect to acute modulation of ion channels. This article briefly summarizes stimulus-induced endocytic regulation of major voltage-dependent potassium channels in the heart. The stimuli known to cause their endocytosis include receptor activation, drugs, and low extracellular [K(+)], following which the potassium channels undergo either clathrin-mediated or caveolin-mediated endocytosis. Receptor-mediated endocytic regulation has been demonstrated for Kv1.2, Kv1.5, KCNQ1 (Kv7.1), and Kv4.3, while drug-induced endocytosis has been demonstrated for Kv1.5 and hERG. Low [K(+)](o)-induced endocytosis might be unique for hERG channels, whose electrophysiological characteristics are known to be under strong influence of [K(+)](o). Although the precise mechanisms have not been elucidated, it is obvious that major cardiac voltage-dependent potassium channels are modulated by endocytosis, which leads to changes in cardiac excitability.

  11. Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic beta-cells.

    Science.gov (United States)

    Yoshida, Masashi; Nakata, Masanori; Yamato, Shiho; Dezaki, Katsuya; Sugawara, Hitoshi; Ishikawa, San-e; Kawakami, Masanobu; Yada, Toshihiko; Kakei, Masafumi

    2010-05-28

    Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic beta-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic beta-cells, suppression of glucose metabolism with low glucose concentrations of 2.8mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to -10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0mM but not 10mM MgATP modulated the channel activity in a manner similar to that in beta-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in beta-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.

  12. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  13. Blockade of the voltage-dependent sodium current in isolated rat hippocampal neurons by tetrodotoxin and lidocaine.

    Science.gov (United States)

    Kaneda, M; Oyama, Y; Ikemoto, Y; Akaike, N

    1989-04-10

    The effects of tetrodotoxin and lidocaine on the voltage-dependent sodium current (INa) were studied in the CA1 pyramidal neurons isolated acutely from rat hippocampus using a 'concentration-clamp' technique which combines the intracellular perfusion with a rapid external solution change within a few ms. Tetrodotoxin (TTX) exerted its inhibitory action in time- and dose-dependent manner on the peak amplitude of INa without any apparent effects on both the current activation and inactivation processes of the current. The time course for reaching a steady-state of the inhibitory action shortened with increasing TTX concentration, but the time course of recovery from the inhibition after washing out the toxin was quite the same at any concentrations used. Lidocaine also inhibited dose-dependently the INa, though with slightly accelerating both the activation and inactivation processes. The time courses for reaching the steady-state inhibition and the recovery from the inhibition were much shorter than those in the case of TTX. The results indicate that the voltage-dependent sodium channel of mammalian brain neuron is TTX-sensitive as well as that of peripheral neuron and that the mode of TTX inhibition on the INa is quite different from that of lidocaine.

  14. Eugenol dilates rat cerebral arteries by inhibiting smooth muscle cell voltage-dependent calcium channels.

    Science.gov (United States)

    Peixoto-Neves, Dieniffer; Leal-Cardoso, Jose Henrique; Jaggar, Jonathan H

    2014-11-01

    Plants high in eugenol, a phenylpropanoid compound, are used as folk medicines to alleviate diseases including hypertension. Eugenol has been demonstrated to relax conduit and ear arteries and reduce systemic blood pressure, but mechanisms involved are unclear. Here, we studied eugenol regulation of resistance-size cerebral arteries that control regional brain blood pressure and flow and investigated mechanisms involved. We demonstrate that eugenol dilates arteries constricted by either pressure or membrane depolarization (60 mM K) in a concentration-dependent manner. Experiments performed using patch-clamp electrophysiology demonstrated that eugenol inhibited voltage-dependent calcium (Ca) currents, when using Ba as a charge carrier, in isolated cerebral artery smooth muscle cells. Eugenol inhibition of voltage-dependent Ca currents involved pore block, a hyperpolarizing shift (∼-10 mV) in voltage-dependent inactivation, an increase in the proportion of steady-state inactivating current, and acceleration of inactivation rate. In summary, our data indicate that eugenol dilates cerebral arteries by means of multimodal inhibition of voltage-dependent Ca channels.

  15. G protein-induced trafficking of voltage-dependent calcium channels.

    Science.gov (United States)

    Tombler, Eugene; Cabanilla, Nory Jun; Carman, Paul; Permaul, Natasha; Hall, John J; Richman, Ryan W; Lee, Jessica; Rodriguez, Jennifer; Felsenfeld, Dan P; Hennigan, Robert F; Diversé-Pierluissi, María A

    2006-01-20

    Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.

  16. Lavender Oil-Potent Anxiolytic Properties via Modulating Voltage Dependent Calcium Channels

    OpenAIRE

    2013-01-01

    Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, bioc...

  17. Scorpion toxin prolongs an inactivation phase of the voltage-dependent sodium current in rat isolated single hippocampal neurons.

    Science.gov (United States)

    Kaneda, M; Oyama, Y; Ikemoto, Y; Akaike, N

    1989-05-15

    The effects of scorpion toxin on the voltage-dependent sodium current (INa) of CA1 pyramidal neurons isolated from rat hippocampus were studied under the single-electrode voltage-clamp condition using a 'concentration-clamp' technique. The toxin increased the peak amplitude of INa and prolonged its inactivation phase in a time- and dose-dependent manner. Inactivation phase of INa proceeded with two exponential components in the absence (control) and presence of the toxin. In the toxin-treated neurons, both the time constant of slow component and its fractional contribution to the total current increased dose-dependently while the fractional contribution of the fast one decreased in a dose-dependent fashion without changing its time constant. Actions of scorpion toxin on the sodium channels of hippocampal pyramidal neurons were essentially similar to those of peripheral preparations. Therefore, it can be concluded that the sodium channels of mammalian brain neurons have structures and functions similar to peripheral channels.

  18. Voltage-dependent Ca2+ channel and Na+ channel in frog taste cells.

    Science.gov (United States)

    Kashiwayanagi, M; Miyake, M; Kurihara, K

    1983-01-01

    Frog taste cells were hyperpolarized by injecting an inward current pulse, and regenerative anode-break potentials were observed at the termination of the current pulse. The results obtained are as follows. 1) The magnitude of the anode-break potentials increased with the extent of hyperpolarization of taste cells and reached a saturation level around -200 mV. 2) The magnitudes of the anode-break potentials observed in 80 different taste cells hyperpolarized to about -200 mV were distributed widely from cell to cell. The average magnitude was 39 mV. 3) The anode-break potentials were recorded after the lingual artery was perfused with artificial solutions containing various channel blockers. The results indicated that the anode-break potentials are composed of Na+ and Ca2+ components. 4) The slope of the current-voltage relation obtained with cells hyperpolarized to 100 mV was appreciably decreased above -50 mV by application of tetrodotoxin to the perfusing solution. Discussion was made on possible roles of the voltage-dependent Na+ and Ca2+ channels in the electrotonic spreading of the depolarization at the receptor membranes to the synaptic area and in releasing a chemical transmitter.

  19. KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain*

    OpenAIRE

    2010-01-01

    Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5L) and S6 C terminus (S6T). From these data, we hypothesiz...

  20. Voltage dependence of rate functions for Na+ channel inactivation within a membrane

    CERN Document Server

    Vaccaro, Samuel R

    2015-01-01

    The inactivation of a Na+ channel occurs when the activation of the charged S4 segment of domain IV, with rate functions $\\alpha_{i}$ and $\\beta_{i}$, is followed by the binding of an intracellular hydrophobic motif which blocks conduction through the ion pore, with rate functions $\\gamma_{i}$ and $\\delta_{i}$. During a voltage clamp of the Na+ channel, the solution of the master equation for inactivation reduces to the relaxation of a rate equation when the binding of the inactivation motif is rate limiting ($\\alpha_{i} \\gg \\gamma_{i}$ and $\\beta_{i} \\gg \\delta_{i}$). The voltage dependence of the derived forward rate function for Na+ channel inactivation has an exponential dependence on the membrane potential for small depolarizations and approaches a constant value for larger depolarizations, whereas the voltage dependence of the backward rate function is exponential, and each rate has a similar form to the Hodgkin-Huxley empirical rate functions for Na+ channel inactivation in the squid axon.

  1. The Eag domain regulates the voltage-dependent inactivation of rat Eag1 K+ channels.

    Directory of Open Access Journals (Sweden)

    Ting-Feng Lin

    Full Text Available Eag (Kv10 and Erg (Kv11 belong to two distinct subfamilies of the ether-à-go-go K+ channel family (KCNH. While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1 and human Erg (hERG1 channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4-S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K+ channels.

  2. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    Science.gov (United States)

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

  3. Selective modulation of cellular voltage dependent calcium channels by hyperbaric pressure - a suggested HPNS partial mechanism

    Directory of Open Access Journals (Sweden)

    Ben eAviner

    2014-05-01

    Full Text Available Professional deep sea divers experience motor and cognitive impairment, known as High Pressure Neurological Syndrome (HPNS, when exposed to pressures of 100 msw (1.1MPa and above, considered to be the result of synaptic transmission alteration. Previous studies have indicated modulation of presynaptic Ca2+ currents at high pressure. We directly measured for the first time pressure effects on the currents of voltage dependent Ca2+ channels (VDCCs expressed in Xenopus oocytes. Pressure selectivity augmented the current in CaV1.2 and depressed it in CaV3.2 channels. Pressure application also affected the channels' kinetics, such as ƮRise, ƮDecay. Pressure modulation of VDCCs seems to play an important role in generation of HPNS signs and symptoms.

  4. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating

    Directory of Open Access Journals (Sweden)

    Mark Alan Zaydman

    2014-05-01

    Full Text Available Voltage-gated potassium (Kv channels contain voltage-sensing (VSD and pore-gate (PGD structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7 channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2. Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

  5. PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating.

    Science.gov (United States)

    Zaydman, Mark A; Cui, Jianmin

    2014-01-01

    Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

  6. The effects of S4 segments on the voltage-dependence of inactivation for Cav3.1 calcium channels

    Institute of Scientific and Technical Information of China (English)

    LI JunYing

    2007-01-01

    T-type calcium channels exhibit fast voltage-dependent inactivation,for which the underlying structure-function relationship still remains unclear.To investigate the roles of S4 segments in voltage-dependent inactivation of T-type calcium channels,we created S4 replacement chimeras between Cav3.1 calcium channels(fast voltage-dependent inactivation)and Cav1.2 calcium channels(little oltage-dependent inactivation)by replacing S4s in Cav3.1 with the corresponding regions in Cav1.2.Wild type and chimeric channels were expressed in Xenopus oocytes and channel currents were recorded with two-electrode voltage-clamp.We showed that replacing S4 region in domain I shifted voltage-dependence for inactivation of Cav3.1 to the left,and the V0.5 inact and kinact value were significantly changed.However replacing S4s in domains Ⅱ-Ⅳ had no effects on the voltage-dependent inactivation properties.These results suggest that the roles of S4 segments in domains Ⅰ-Ⅳ are different,and S4 in domain I is likely to be involved in voltage-dependent Inactivation process.Its movement during membrane depolarization may trigger a conformational change in the inactivation gate.

  7. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  8. Cortisone dissociates voltage-dependent K+ channel from its beta subunit

    Science.gov (United States)

    Pan, Yaping; Weng, Jun; Kabaleeswaran, Venkataraman; Li, Huiguang; Cao, Yu; Bhosle, Rahul C.; Zhou, Ming

    2009-01-01

    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with beta subunits (Kvβ) and certain Kvβs, for example Kvβ1, have an N-terminal segment that closes a channel by the N-type inactivation mechanism. In principle dissociation of Kvβ1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases mammalian (rat) Kv1 channel activity by binding to Kvβ1. A crystal structure of the Kvβ-cortisone complex was solved to 1.82 Å resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvβ. The new mode of channel modulation may be explored by native or synthetic ligands to fine tune cellular excitability. PMID:18806782

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  10. KCNQ1 channels voltage dependence through a voltage-dependent binding of the S4-S5 linker to the pore domain.

    Science.gov (United States)

    Choveau, Frank S; Rodriguez, Nicolas; Abderemane Ali, Fayal; Labro, Alain J; Rose, Thierry; Dahimène, Shehrazade; Boudin, Hélène; Le Hénaff, Carole; Escande, Denis; Snyders, Dirk J; Charpentier, Flavien; Mérot, Jean; Baró, Isabelle; Loussouarn, Gildas

    2011-01-07

    Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

  11. State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

    OpenAIRE

    Miranda, Pablo; Contreras, Jorge E.; Plested, Andrew J. R.; Sigworth, Fred J.; Holmgren, Miguel; Giraldez, Teresa

    2013-01-01

    Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductan...

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

    Institute of Scientific and Technical Information of China (English)

    LI Lu; LIU Jihong; LI Jiagui; YE Zhangqun

    2006-01-01

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

  13. Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect.

    Science.gov (United States)

    Lemasters, John J; Holmuhamedov, Ekhson L; Czerny, Christoph; Zhong, Zhi; Maldonado, Eduardo N

    2012-06-01

    Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

  14. Over Expression of Voltage Dependent Anion Channel 2 (VDAC2 in Muscles of Electrically Stunned Chickens

    Directory of Open Access Journals (Sweden)

    Norshahida Abu Samah, Azura Amid, and Faridah Yusof

    2011-12-01

    Full Text Available Water bath stunning is a common practice in commercial slaughterhouses. Such treatment is economic and in line with animal welfare practice. However, the conditions applied for the stunning process may vary from a slaughterhouse to another slaughterhouse. Such a loose regulation on the stunning procedure has opened up doors for food adulteration such as over dose stunning. In this study, a simple and reliable approach using proteomics have been developed to study the effect of different currents and voltages in stunning on the protein expression of the chickens. Protein profiles of the chickens were constructed in order to detect any differences in protein expression and modifications. The different voltage studied were 10 V, 40 V and 70 V while the values for current studied were 0.25 A, 0.5 A, and 0.75 A. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption ionization- time of flight (MALDI TOF spectrometry identification, Voltage dependent anion channel 2 (VDAC2 was identified to be over expressed in the muscle sample of over stunned chicken. The over expression of VDAC2 was confirmed at the transcriptional level of RNA expression. Real Time PCR showed that all over stunned samples contained higher mRNA expression level for VDAC2 genes. The mRNA level of VDAC2 was up-regulated by 59.87 fold change when normalized with housekeeping gene. In conclusion, VDAC2 could serve as potential biomarkers for identification of electrically stimulated chickens. The existence of these biomarkers will help to monitor the slaughtering and stunning process in the future. It will revolutionize the food authentication field and give a new breathe to the meat industry.ABSTRAK: Kaedah "waterbath stunning" merupakan amalan biasa di pusat-pusat penyembelihan. Kaedah ini adalah ekonomik dan selari dengan amalan kebajikan haiwan. Walaubagaimanapun, syarat-syarat yang digunakan untuk proses kejutan tersebut mungkin

  15. Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels.

    Science.gov (United States)

    Zhang, Yun; Chen, Yu-Hang; Bangaru, Saroja D; He, Linling; Abele, Kathryn; Tanabe, Shihori; Kozasa, Tohru; Yang, Jian

    2008-12-24

    G-protein (Gbetagamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca(2+) channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gbetagamma from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca(2+) channel beta subunit (Ca(v)beta) and a rigid alpha-helical structure between the alpha-interacting domain (AID), the primary Ca(v)beta docking site on the channel alpha(1) subunit, and the pore-lining IS6 segment. Gbetagamma inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca(v)beta were produced by washing out a mutant Ca(v)beta with a reduced affinity for the AID. These beta-less channels were still inhibited by Gbetagamma, but without any voltage dependence, indicating that Ca(v)beta is indispensable for voltage-dependent Gbetagamma inhibition. A truncated Ca(v)beta containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gbetagamma inhibition. Gbetagamma did not alter inactivation properties, and channels recovered from Gbetagamma inhibition exhibited the same activation property as un-inhibited channels, indicating that Gbetagamma does not dislodge Ca(v)beta from the inhibited channel. Furthermore, voltage-dependent Gbetagamma inhibition was abolished when the rigid alpha-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca(v)beta regulation of channel gating, revealing a pivotal role of this rigid alpha-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gbetagamma-binding pocket through a rigid alpha-helix induced partly by the Ca(v)beta GK domain, causes the

  16. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    Directory of Open Access Journals (Sweden)

    Dong-Hai Liu

    Full Text Available Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  17. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    Science.gov (United States)

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  18. A voltage-dependent persistent sodium current in mammalian hippocampal neurons.

    Science.gov (United States)

    French, C R; Sah, P; Buckett, K J; Gage, P W

    1990-06-01

    Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at

  19. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

    Science.gov (United States)

    Yagoda, Nicholas; von Rechenberg, Moritz; Zaganjor, Elma; Bauer, Andras J; Yang, Wan Seok; Fridman, Daniel J; Wolpaw, Adam J; Smukste, Inese; Peltier, John M; Boniface, J Jay; Smith, Richard; Lessnick, Stephen L; Sahasrabudhe, Sudhir; Stockwell, Brent R

    2007-06-14

    Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  1. Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels.

    Science.gov (United States)

    Schuwald, Anita M; Nöldner, Michael; Wilmes, Thomas; Klugbauer, Norbert; Leuner, Kristina; Müller, Walter E

    2013-01-01

    Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

  2. Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels.

    Directory of Open Access Journals (Sweden)

    Anita M Schuwald

    Full Text Available Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

  3. Reversal of HCN channel voltage dependence via bridging of the S4-S5 linker and Post-S6.

    Science.gov (United States)

    Prole, David L; Yellen, Gary

    2006-09-01

    Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4-S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands.

  4. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  5. Altered ischemic cerebral injury in mice lacking αIE subunit of the voltage-dependent Ca2+ channel

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective ①To set up a stable and reproducible focal cerebral infarct modelin mice; (②To examine theinvolvement of αIE subunit of voltage-dependent Ca2 + channel in cerebral ischemic injury. Methods Male C57BL/6J Jclmice 8 ~ 12w and F4 ~ F6αIE subunit of Ca2+ channel mutant mice were both used in this study. All animals were allowedto freely access to food and water before and after operation. Animals were anesthetized with pentobarbital sodium 60mg/kg,ip. Rectal temperature was continuously monitored before, during and after operation, and maintained at (36.6 +0.1 )°C by a autoregulating pad. To produce pilot models, the middle cerebral artery (MCA) was occluded either by sur-gical ligation or electrical coagulation and in some models the common carotid artery (CCA) was surgically ligated in tan-dem. In our latter work the MCA was cut off soon after it was ligated or coagulated in order to make sure that the bloodflow was occluded completely. The MCA was coagulated or ligated with a bipolar coagulator or microsurgery suture at thesite just superior to the rhinal fissure. Twenty~four hours after the operation, the mice were anesthetized and decapitated,then their brains were dissected from the skull and put into cold artificial brain spinal fluid as soon as possible. Lmm thickcoronal sections were cut by vibratome and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for30min. Every section was photographed positively and the whole infarction volume was calculated by summing up the in-farction volumes of all sections by NIH Image System. Infarction ratio ( % ) was also calculated by the following fommula:(contralateral volume-ipsilateral undamaged volume)/contralateral volume × 100% to eliminate the influence of edema.In brief, the mutant mice were produced with gene targeting technique. F4 ~ F6 mice were used in this experiment. Alloffsprings were genotyped by the polymerase chain reaction (PCR) and the genotypes remained umknown

  6. Voltage dependence of Hodgkin-Huxley rate functions for a multistage K+ channel voltage sensor within a membrane

    Science.gov (United States)

    Vaccaro, S. R.

    2014-11-01

    The activation of a K+channel sensor in two sequential stages during a voltage clamp may be described as the translocation of a Brownian particle in an energy landscape with two large barriers between states. A solution of the Smoluchowski equation for a square-well approximation to the potential function of the S4 voltage sensor satisfies a master equation and has two frequencies that may be determined from the forward and backward rate functions. When the higher-frequency terms have small amplitude, the solution reduces to the relaxation of a rate equation, where the derived two-state rate functions are dependent on the relative magnitude of the forward rates (α and γ ) and the backward rates (β and δ ) for each stage. In particular, the voltage dependence of the Hodgkin-Huxley rate functions for a K+channel may be derived by assuming that the rate functions of the first stage are large relative to those of the second stage—α ≫γ and β ≫δ . For a Shaker IR K+ channel, the first forward and backward transitions are rate limiting (α <γ and δ ≪β ), and for an activation process with either two or three stages, the derived two-state rate functions also have a voltage dependence that is of a similar form to that determined for the squid axon. The potential variation generated by the interaction between a two-stage K+ ion channel and a noninactivating Na+ ion channel is determined by the master equation for K+channel activation and the ionic current equation when the Na+channel activation time is small, and if β ≪δ and α ≪γ , the system may exhibit a small amplitude oscillation between spikes, or mixed-mode oscillation, in which the slow closed state modulates the K+ ion channel conductance in the membrane.

  7. Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Liu-Min Fan

    2009-01-01

    Free cytosolic Ca~(2+) ([Ca~(2+)]_(cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca~(2+)]_(cyt) elevation is associated with Ca~(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca~(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca~(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba~(2+) and Ca~(2+), and their activities can be inhibited by micromolar Gd~(3+). The unitary conductance and the reversal potential of the channels depend on the Ca~(2+) or Ba~(2+) gradients across the plasma membrane. The inward whole-cell Ca~(2+) (Ba~(2+)) current, as well as the unitary current amplitude and NP. of the single Ca~(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NP_o of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

  8. Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1.

    Science.gov (United States)

    Elinder, Fredrik; Madeja, Michael; Zeberg, Hugo; Århem, Peter

    2016-10-18

    The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr(2+)) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr(2+) (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr(2+)-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr(2+) acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

  9. Correlation character of ionic current fluctuations: analysis of ion current through a voltage-dependent potassium single channel.

    Science.gov (United States)

    Tong-Han, Lan; Huang, Xi; Jia-Rui, Lin

    2005-10-03

    The gating of ion channels has widely been modeled by assuming the transition between open and closed states is a memoryless process. Nevertheless, the statistical analysis of an ionic current signal recorded from voltage dependence K(+) single channel is presented. Calculating the sample auto-correlation function of the ionic current based on the digitized signals, rather than the sequence of open and closed states duration time. The results provide evidence for the existence of memory. For different voltages, the ion channel current fluctuation has different correlation attributions. The correlations in data generated by simulation of two Markov models, on one hand, auto-correlation function of the ionic current shows a weaker memory, after a delayed period of time, the attribute of memory does not exist; on the other hand, the correlation depends on the number of states in the Markov model. For V(p)=-60 mV pipette potential, spectral analysis of ion channel current was conducted, the result indicates that the spectrum is not a flat spectrum, the data set from ionic current fluctuations shows considerable variability with a broad 1/f -like spectrum, alpha=1.261+/-0.24. Thus the ion current fluctuations give information about the kinetics of the channel protein, the results suggest the correlation character of ion channel protein nonlinear kinetics regardless of whether the channel is in open or closed state.

  10. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D;

    2001-01-01

    .2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed......The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1...

  11. Characterization and functional analysis of voltage-dependent anion channel 1 (VDAC1) from orange-spotted grouper (Epinephelus coioides).

    Science.gov (United States)

    Shi, Yan; Zhao, Zhe; Hong, Xiaoyou; Chen, Kunci; Zhu, Xinping

    2014-07-01

    The voltage-dependent anion channel (VDAC) is a highly conserved integral protein of mitochondria in different eukaryotic species. It forms a selective channel in the mitochondrial outer membrane that serves as the controlled pathway for small metabolites and ions. In this study, a VDAC gene, EcVDAC1, was isolated from orange-spotted grouper (Epinephelus coioides). The EcVDAC1 exhibits ubiquitous expression in various tissues of orange-spotted grouper and is upregulated in liver, gill, and spleen after stimulation with lipopolysaccharides (LPS). Subcellular localization analysis shows that the EcVDAC1 protein colocalized with the mitochondria. A caspase-3 assay demonstrates that overexpression of the EcVDAC1 induced apoptotic cell death in fathead minnow cells. The data presented in this study provide new information regarding the relationship between LPS and the EcVDAC1 gene, suggesting that the fish VDAC1 gene may play an important role in antibacterial immune response.

  12. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

    Indian Academy of Sciences (India)

    HAN SOL KIM; HONGLIANG LI; HYE WON KIM; SUNG EUN SHIN; IL-WHAN CHOI; AMY L FIRTH; HYOWEON BANG; YOUNG MIN BAE; WON SUN PARK

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv)channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertralinedecreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 μM and a slope value (Hillcoefficient) of 0.61. Although the application of 1 μM sertraline did not affect the steady-state activation curves,sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine,had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From theseresults, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptakeinhibition by shifting inactivation curves to a more negative potential.

  13. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

    Science.gov (United States)

    Kim, Han Sol; Li, Hongliang; Kim, Hye Won; Shin, Sung Eun; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Bae, Young Min; Park, Won Sun

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertraline decreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 mu M and a slope value (Hill coefficient) of 0.61. Although the application of 1 mu M sertraline did not affect the steady-state activation curves, sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine, had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From these results, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptake inhibition by shifting inactivation curves to a more negative potential.

  14. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2001-01-01

    The distribution of voltage-dependent calcium channels in kidney pre- and postglomerular resistance vessels was determined at the molecular and functional levels. Reverse transcription-polymerase chain reaction analysis of microdissected rat preglomerular vessels and cultured smooth muscle cells...... showed coexpression of mRNAs for T-type subunits (Ca(V)3.1, Ca(V)3.2) and for an L-type subunit (Ca(V)1.2). The same expression pattern was observed in juxtamedullary efferent arterioles and outer medullary vasa recta. No calcium channel messages were detected in cortical efferent arterioles. Ca(V)1.......2 protein was demonstrated by immunochemical labeling of rat preglomerular vasculature and juxtamedullary efferent arterioles and vasa recta. Cortical efferent arterioles were not immunopositive. Recordings of intracellular calcium concentration with digital fluorescence imaging microscopy showed...

  15. Functional coupling between large-conductance potassium channels and Cav3.2 voltage-dependent calcium channels participates in prostate cancer cell growth

    Directory of Open Access Journals (Sweden)

    Florian Gackière

    2013-07-01

    It is strongly suspected that potassium (K+ channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K+ channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K+ current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K+ channel family involved in setting the resting membrane potential in LNCaP cells at around −40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.

  16. Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

    Science.gov (United States)

    Picher, M; Decrouy, A; Rousseau, E

    1996-02-21

    Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-10-10

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

  19. Aconitum sp. alkaloids: The modulation of voltage-dependent Na+channels, toxicity and antinociceptive properties

    NARCIS (Netherlands)

    Friese, Jutta; Gleitz, Johannes; Gutser, Ulrike T.; Heubach, Jürgen F.; Matthiesen, Theo; Wilffert, Bob; Selve, Norma

    1997-01-01

    Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+channel epitope site 2, (2) alterations in synaptosoml Na+and Ca2+concentration ([Na+](i), [Ca2+](i)), (3) arrhyth

  20. The sea anemone Bunodosoma caissarum toxin BcIII modulates the sodium current kinetics of rat dorsal root ganglia neurons and is displaced in a voltage-dependent manner.

    Science.gov (United States)

    Salceda, Emilio; López, Omar; Zaharenko, André J; Garateix, Anoland; Soto, Enrique

    2010-03-01

    Sea anemone toxins bind to site 3 of the sodium channels, which is partially formed by the extracellular linker connecting S3 and S4 segments of domain IV, slowing down the inactivation process. In this work we have characterized the actions of BcIII, a sea anemone polypeptide toxin isolated from Bunodosoma caissarum, on neuronal sodium currents using the patch clamp technique. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study (n=65). The main effects of BcIII were a concentration-dependent increase in the sodium current inactivation time course (IC(50)=2.8 microM) as well as an increase in the current peak amplitude. BcIII did not modify the voltage at which 50% of the channels are activated or inactivated, nor the reversal potential of sodium current. BcIII shows a voltage-dependent action. A progressive acceleration of sodium current fast inactivation with longer conditioning pulses was observed, which was steeper as more depolarizing were the prepulses. The same was observed for other two anemone toxins (CgNa, from Condylactis gigantea and ATX-II, from Anemonia viridis). These results suggest that the binding affinity of sea anemone toxins may be reduced in a voltage-dependent manner, as has been described for alpha-scorpion toxins.

  1. Voltage dependence of Hodgkin-Huxley rate functions for a multistage K^{+} channel voltage sensor within a membrane.

    Science.gov (United States)

    Vaccaro, S R

    2014-11-01

    The activation of a K^{+} channel sensor in two sequential stages during a voltage clamp may be described as the translocation of a Brownian particle in an energy landscape with two large barriers between states. A solution of the Smoluchowski equation for a square-well approximation to the potential function of the S4 voltage sensor satisfies a master equation and has two frequencies that may be determined from the forward and backward rate functions. When the higher-frequency terms have small amplitude, the solution reduces to the relaxation of a rate equation, where the derived two-state rate functions are dependent on the relative magnitude of the forward rates (α and γ) and the backward rates (β and δ) for each stage. In particular, the voltage dependence of the Hodgkin-Huxley rate functions for a K^{+} channel may be derived by assuming that the rate functions of the first stage are large relative to those of the second stage-α≫γ and β≫δ. For a Shaker IR K^{+} channel, the first forward and backward transitions are rate limiting (αchannel and a noninactivating Na^{+} ion channel is determined by the master equation for K^{+} channel activation and the ionic current equation when the Na^{+} channel activation time is small, and if β≪δ and α≪γ, the system may exhibit a small amplitude oscillation between spikes, or mixed-mode oscillation, in which the slow closed state modulates the K^{+} ion channel conductance in the membrane.

  2. trans-Caryophyllene, a Natural Sesquiterpene, Causes Tracheal Smooth Muscle Relaxation through Blockade of Voltage-Dependent Ca2+ Channels

    Directory of Open Access Journals (Sweden)

    Jader Santos Cruz

    2012-10-01

    Full Text Available trans-Caryophyllene is a major component in the essential oils of various species of medicinal plants used in popular medicine in Brazil. It belongs to the chemical class of the sesquiterpenes and has been the subject of a number of studies. Here, we evaluated the effects of this compound in airway smooth muscle. The biological activities of trans-caryophyllene were examined in isolated bath organs to investigate the effect in basal tonus. Electromechanical and pharmacomechanical couplings were evaluated through the responses to K+ depolarization and exposure to acetylcholine (ACh, respectively. Isolated cells of rat tracheal smooth muscle were used to investigate trans-caryophyllene effects on voltage-dependent Ca2+ channels by using the whole-cell voltage-clamp configuration of the patch-clamp technique. trans-Caryophyllene showed more efficiency in the blockade of electromechanical excitation-contraction coupling while it has only minor inhibitory effect on pharmacomechanical coupling. Epithelium removal does not modify tracheal smooth muscle response elicited by trans-caryophyllene in the pharmacomechanical coupling. Under Ca2+-free conditions, pre-exposure to trans-caryophyllene did not reduce the contraction induced by ACh in isolated rat tracheal smooth muscle, regardless of the presence of intact epithelium. In the whole-cell configuration, trans-caryophyllene (3 mM, inhibited the inward Ba2+ current (IBa to approximately 50% of control levels. Altogether, our results demonstrate that trans-caryophyllene has anti-spasmodic activity on rat tracheal smooth muscle which could be explained, at least in part, by the voltage-dependent Ca2+ channels blockade.

  3. Voltage-dependent K channels in protoplasts of trap-lobe cells of Dionaea muscipula.

    Science.gov (United States)

    Iijima, T; Hagiwara, S

    1987-01-01

    The outward rectification of the K+ current in mesophyll cell protoplasts from trap-lobes of Dionaea muscipula was studied with the patch-clamp technique. The rectification had instantaneous and time-dependent components. Changes in [K+]i strongly affected the conductance voltage relation of the plasma membrane while changes in [K+]o had little effect on the relation. Thus, the outward rectification depends on the membrane voltage and the concentration of intracellular K+. Corresponding single-channel activities were observed both in the intact membrane (cell-attached recording) and in excised patches. The single-channel conductance was about 3.3 pS with symmetrical solutions containing 30 mM K+.

  4. Stochastic Dynamics of Electrical Membrane with Voltage-Dependent Ion Channel Fluctuations

    CERN Document Server

    Qian, Hong; Qian, Min

    2014-01-01

    Brownian ratchet like stochastic theory for the electrochemical membrane system of Hodgkin-Huxley (HH) is developed. The system is characterized by a continuous variable $Q_m(t)$, representing mobile membrane charge density, and a discrete variable $K_t$ representing ion channel conformational dynamics. A Nernst-Planck-Nyquist-Johnson type equilibrium is obtained when multiple conducting ions have a common reversal potential. Detailed balance yields a previously unknown relation between the channel switching rates and membrane capacitance, bypassing Eyring-type explicit treatment of gating charge kinetics. From a molecular structural standpoint, membrane charge $Q_m$ is a more natural dynamic variable than potential $V_m$; our formalism treats $Q_m$-dependent conformational transition rates $\\lambda_{ij}$ as intrinsic parameters. Therefore in principle, $\\lambda_{ij}$ vs. $V_m$ is experimental protocol dependent,e.g., different from voltage or charge clamping measurements. For constant membrane capacitance pe...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  6. Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels.

    Science.gov (United States)

    Cens, Thierry; Rousset, Matthieu; Collet, Claude; Charreton, Mercedes; Garnery, Lionel; Le Conte, Yves; Chahine, Mohamed; Sandoz, Jean-Christophe; Charnet, Pierre

    2015-03-01

    Voltage-gated Ca(2+) channels allow the influx of Ca(2+) ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca(2+) transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca(2+) channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca(2+) channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca(2+) channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca(2+) channels.

  7. Regulation of sodium channel function by bilayer elasticity

    DEFF Research Database (Denmark)

    Lundbaek, Jens A; Birn, Pia; Hansen, Anker J

    2004-01-01

    be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100......, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change...... in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein...

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  10. Lidocaine block of cardiac sodium channels

    OpenAIRE

    Bean, BP; Cohen, CJ; Tsien, RW

    1983-01-01

    Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding pote...

  11. Multiphasic profiles for voltage-dependent K+ channels: Reanalysis of data of MacKinnon and coworkers

    CERN Document Server

    Nissen, Per

    2016-01-01

    In a study of the role that voltage-dependent K+ channels may have in the mechanosensation of living cells (Schmidt et al. Proc Soc Natl Acad Sci USA 109: 10352-10357. 2012), the data were as conventionally done fitted by a Boltzmann function. However, as also found for other data for ion channels, this interpretation must be rejected in favor of a multiphasic profile, a series of straight lines separated by discontinuous transitions, quite often in the form of noncontiguities (jumps). The data points in the present study are often very unevenly distributed around the curvilinear profiles. Thus, for 43 of the 75 profiles, the probability is less than 5% that the uneven distribution is due to chance, for 26 the probability is less than 1%, and for 12 the probability is less than 0.1%, giving a vanishingly low overall probability for all profiles. Especially at low voltages, the differences between the fits to curvilinear and multiphasic profiles may be huge. In the multiphasic profiles, adjacent lines are quit...

  12. Lack of negatively charged residues at the external mouth of Kir2.2 channels enable the voltage-dependent block by external Mg2+.

    Directory of Open Access Journals (Sweden)

    Junwei Li

    Full Text Available Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.

  13. VOLTAGE-DEPENDENT SODIUM AND POTASSIUM, BUT NO CALCIUM CONDUCTANCES IN DDT1 MF-2 SMOOTH-MUSCLE CELLS

    NARCIS (Netherlands)

    MOLLEMAN, A; NELEMANS, A; VANDENAKKER, J; DUIN, M; DENHERTOG, A

    1991-01-01

    Voltage-dependent inward and outward membrane currents were investigated in the DDT1 MF-2 smooth muscle cell line using the whole-cell patch-clamp technique. Application of a pulse protocol with subsequent depolarizing voltage steps elicited an inactivating inward current and a non-inactivating outw

  14. Voltage-Dependent Anion Channel 1(VDAC1) Participates the Apoptosis of the Mitochondrial Dysfunction in Desminopathy

    Science.gov (United States)

    Mo, Yanqing; Gong, Qi; Jiang, Aihua; Zhao, Jing

    2016-01-01

    Desminopathies caused by the mutation in the gene coding for desmin are genetically protein aggregation myopathies. Mitochondrial dysfunction is one of pathological changes in the desminopathies at the earliest stage. The molecular mechanisms of mitochondria dysfunction in desminopathies remain exclusive. VDAC1 regulates mitochondrial uptake across the outer membrane and mitochondrial outer membrane permeabilization (MOMP). Relationships between desminopathies and Voltage-dependent anion channel 1 (VDAC1) remain unclear. Here we successfully constructed the desminopathy rat model, evaluated with conventional stains, containing hematoxylin and eosin (HE), Gomori Trichrome (MGT), (PAS), red oil (ORO), NADH-TR, SDH staining and immunohistochemistry. Immunofluorescence results showed that VDAC1 was accumulated in the desmin highly stained area of muscle fibers of desminopathy patients or desminopathy rat model compared to the normal ones. Meanwhile apoptosis related proteins bax and ATF2 were involved in desminopathy patients and desminopathy rat model, but not bcl-2, bcl-xl or HK2.VDAC1 and desmin are closely relevant in the tissue splices of deminopathies patients and rats with desminopathy at protein lever. Moreover, apoptotic proteins are also involved in the desminopathies, like bax, ATF2, but not bcl-2, bcl-xl or HK2. This pathological analysis presents the correlation between VDAC1 and desmin, and apoptosis related proteins are correlated in the desminopathy. Furthermore, we provide a rat model of desminopathy for the investigation of desmin related myopathy. PMID:27941998

  15. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.

    Science.gov (United States)

    Maldonado, Eduardo N; Sheldon, Kely L; DeHart, David N; Patnaik, Jyoti; Manevich, Yefim; Townsend, Danyelle M; Bezrukov, Sergey M; Rostovtseva, Tatiana K; Lemasters, John J

    2013-04-26

    Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.

  16. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

  17. Voltage-dependent ionic channels in differentiating neural precursor cells collected from adult mouse brains six hours post-mortem.

    Science.gov (United States)

    Bellardita, Carmelo; Bolzoni, Francesco; Sorosina, Melissa; Marfia, Giovanni; Carelli, Stephana; Gorio, Alfredo; Formenti, Alessandro

    2012-04-01

    A novel type of adult neural precursor cells (NPCs) has been isolated from the subventricular zone of the mouse 6 hr after animal death (T6-NPCs). This condition is supposed to select hypoxia-resistant cells of scientific and clinical interest. Ionic channels are ultimately the expression of the functional maturation of neurons, so the aim of this research was to characterize the pattern of the main voltage-dependent ionic channels in T6-NPCs differentiating to a neuronal phenotype, comparing it with NPCs isolated soon after death (T0-NPCs). T6- and T0-NPCs grow in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Differentiation was performed in small wells without the addition of growth factors, in the presence of adhesion molecules, fetal bovine serum, and leukemia inhibitory factor. Ionic currents, recorded by means of whole-cell patch-clamp, namely, I(Ca2+) HVA, both L- and non-L-type, I(K+) delayed rectifying, I(K+) inward rectifier, transient I(K+A) , and TTX-sensitive I(Na+) have been found, although Na(+) currents were found in only a small percentage of cells and after the fifth week of differentiation. No significant differences in current types, density, orcell capacitance were observed between T6-NPCs and T0-NPCs. The sequence in which the markers appear in new neural cells is not necessarily a fixed program, but the discrepancies in morphological, biochemical, and electrophysiological maturation of mouse NPCs to neurons, possibly different in vivo, suggest that the various steps of the differentiation are independently regulated. Therefore, in addition to morphological and biochemical data, functional tests should be considered for characterizing the maturation of neurons.

  18. The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

    Science.gov (United States)

    Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

    2015-12-25

    The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

  19. Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

    Science.gov (United States)

    Budelier, Melissa M; Cheng, Wayland W L; Bergdoll, Lucie; Chen, Zi-Wei; Janetka, James W; Abramson, Jeff; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F; Whitelegge, Julian P; Evers, Alex S

    2017-06-02

    Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr(83) and Glu(73), respectively. When Glu(73) was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr(62) within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu(73) residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

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

    2014-01-01

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

  1. Ca2+ channel inhibitor NNC 55-0396 inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

    Science.gov (United States)

    Son, Youn Kyoung; Hong, Da Hye; Li, Hongliang; Kim, Dae-Joong; Na, Sung Hun; Park, Hongzoo; Jung, Won-Kyo; Choi, Il-Whan; Park, Won Sun

    2014-01-01

    We demonstrated the inhibitory effect of NNC 55-0396, a T-type Ca(2+) channel inhibitor, on voltage-dependent K(+) (K(V)) channels in freshly isolated rabbit coronary arterial smooth muscle cells. NNC 55-0396 decreased the amplitude of K(V) currents in a concentration-dependent manner, with an IC(50) of 0.080 μM and a Hill coefficient of 0.76.NNC 55-0396 did not affect steady-state activation and inactivation curves, indicating that the compound does not affect the voltage sensitivity of K(V) channel gating. Both the K(V) currents and the inhibitory effect of NNC 55-0396 on K(V) channels were not altered by depletion of extracellular Ca(2+) or intracellular ATP, suggesting that the inhibitory effect of NNC 55-0396 is independent of Ca(2+)-channel activity and phosphorylation-dependent signaling cascades. From these results, we concluded that NNC 55-0396 dosedependently inhibits K(V) currents, independently of Ca(2+)-channel activity and intracellular signaling cascades.

  2. Poly(ethylene glycol-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells.

    Directory of Open Access Journals (Sweden)

    Rikuo Ochi

    Full Text Available Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol cholesteryl ether (PEG-cholesterol is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca(2+ channel currents (I(Ca,L recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of ICa,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-β-cyclodextrin (MβCD is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MβCD:cholesterol complex (cholesterol (MβCD caused inhibition of I(Ca,L but did not augment voltage-dependent inactivation. Incubation with MβCD increased I(Ca,L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MβCD of the cells initially pretreated with PEG-cholesterol, increased I(Ca,L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I(Ca,L more strongly as compared with cholesterol (MβCD. Poly(ethylene glycol conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I(Ca,L.

  3. Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit.

    Science.gov (United States)

    Keum, Dongil; Baek, Christina; Kim, Dong-Il; Kweon, Hae-Jin; Suh, Byung-Chang

    2014-10-01

    G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of

  4. Comparative study of lacosamide and classical sodium channel blocking antiepileptic drugs on sodium channel slow inactivation.

    Science.gov (United States)

    Niespodziany, Isabelle; Leclère, Nathalie; Vandenplas, Catherine; Foerch, Patrik; Wolff, Christian

    2013-03-01

    Many antiepileptic drugs (AEDs) exert their therapeutic activity by modifying the inactivation properties of voltage-gated sodium (Na(v) ) channels. Lacosamide is unique among AEDs in that it selectively enhances the slow inactivation component. Although numerous studies have investigated the effects of AEDs on Na(v) channel inactivation, a direct comparison of results cannot be made because of varying experimental conditions. In this study, the effects of different AEDs on Na(v) channel steady-state slow inactivation were investigated under identical experimental conditions using whole-cell patch-clamp in N1E-115 mouse neuroblastoma cells. All drugs were tested at 100 μM, and results were compared with those from time-matched control groups. Lacosamide significantly shifted the voltage dependence of Na(v) current (I(Na) ) slow inactivation toward more hyperpolarized potentials (by -33 ± 7 mV), whereas the maximal fraction of slow inactivated channels and the curve slope did not differ significantly. Neither SPM6953 (lacosamide inactive enantiomer), nor carbamazepine, nor zonisamide affected the voltage dependence of I(Na) slow inactivation, the maximal fraction of slow inactivated channels, or the curve slope. Phenytoin significantly increased the maximal fraction of slow inactivated channels (by 28% ± 9%) in a voltage-independent manner but did not affect the curve slope. Lamotrigine slightly increased the fraction of inactivated currents (by 15% ± 4%) and widened the range of the slow inactivation voltage dependence. Lamotrigine and rufinamide induced weak, but significant, shifts of I(Na) slow inactivation toward more depolarized potentials. The effects of lacosamide on Na(v) channel slow inactivation corroborate previous observations that lacosamide has a unique mode of action among AEDs that act on Na(v) channels.

  5. Evidence for functional interaction of plasma membrane electron transport, voltage-dependent anion channel and volume-regulated anion channel in frog aorta

    Indian Academy of Sciences (India)

    Rashmi P Rao; J Prakasa Rao

    2010-12-01

    Frog aortic tissue exhibits plasma membrane electron transport (PMET) owing to its ability to reduce ferricyanide even in the presence of mitochondrial poisons, such as cyanide and azide. Exposure to hypotonic solution (108 mOsmol/kg H2O) enhanced the reduction of ferricyanide in excised aortic tissue of frog. Increment in ferricyanide reductase activity was also brought about by the presence of homocysteine (100 M dissolved in isotonic frog Ringer solution), a redox active compound and a potent modulator of PMET. Two plasma-membrane-bound channels, the volume regulated anion channel (VRAC) and the voltage-dependent anion channel (VDAC), are involved in the response to hypotonic stress. The presence of VRAC and VDAC antagonists–tamoxifen, glibenclamide, fluoxetine and verapamil, and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), respectively–inhibited this enhanced activity brought about by either hypotonic stress or homocysteine. The blockers do not affect the ferricyanide reductase activity under isotonic conditions. Taken together, these findings indicate a functional interaction of the three plasma membrane proteins, namely, ferricyanide reductase (PMET), VDAC and VRAC.

  6. Involvement of presynaptic voltage-dependent Kv3 channel in endothelin-1-induced inhibition of noradrenaline release from rat gastric sympathetic nerves.

    Science.gov (United States)

    Nakamura, Kumiko; Shimizu, Takahiro; Tanaka, Kenjiro; Taniuchi, Keisuke; Yokotani, Kunihiko

    2012-11-05

    We previously reported that two types of K(+) channels, the BK type Ca(2+)-activated K(+) channel coupled with phospholipase C (PLC) and the voltage-dependent K(+) channel (Kv channel), are, respectively, involved in the prostanoid TP receptor- and muscarinic M(2) receptor-mediated inhibition of noradrenaline (NA) release from rat gastric sympathetic nerves. In the present study, therefore, we examined whether these K(+) channels are involved in endothelin-1-induced inhibition of NA release, using an isolated, vascularly perfused rat stomach. The gastric sympathetic postganglionic nerves around the left gastric artery were electrically stimulated twice at 2.5 Hz for 1 min, and endothelin-1 was added during the second stimulation. Endothelin-1 (1, 2 and 10 nM) dose-dependently inhibited gastric NA release. Endothelin-1 (2 nM)-induced inhibition of NA release was neither attenuated by PLC inhibitors [U-73122 (3 μM) and ET-18-OCH(3) (3 μM)] nor by Ca(2+)-activated K(+) channel blockers [charybdotoxin (0.1 μM) (a blocker of BK type K(+) channel) and apamin (0.3 μM) (a blocker of SK type K(+) channel)]. The endothelin-1-induced inhibitory response was also not attenuated by α-dendrotoxin (0.1 μM) (a selective inhibitor of Kv1 channel), but abolished by 4-aminopyridine (20 μM) (a selectively inhibitory dose for Kv3 channel). These results suggest the involvement of a voltage-dependent Kv3 channel in the endothelin-1-induced inhibition of NA release from the gastric sympathetic nerves in rats.

  7. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired ß-adrenoceptor-mediated relaxation of renal arteries in hypertension

    DEFF Research Database (Denmark)

    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei;

    2012-01-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown...... strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein...... spontaneously hypertensive rats, which was associated with ˜60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired ß...

  8. Coexpression of voltage-dependent calcium channels Cav1.2, 2.1a, and 2.1b in vascular myocytes

    DEFF Research Database (Denmark)

    Andreasen, Ditte; Friis, Ulla Glenert; Uhrenholt, Torben Rene

    2006-01-01

    , and blocking P-type currents (omega-agatoxin IVA 10 nmol/L) led to 20.2+/-3.0% inhibition, whereas 300 nmol/L of omega agatoxin IVA (blocking P/Q-type) inhibited 45.0+/-7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5+/-6.1% inhibition, simultaneous blockade of L...... microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6+/-3.9% of the voltage-dependent Ca2+ current......-type and P-type channels inhibited 58.0+/-11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7+/-5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse....

  9. Effects of in vitro and in vivo lead exposure on voltage-dependent calcium channels in central neurons of Lymnaea stagnalis.

    Science.gov (United States)

    Audesirk, G

    1987-01-01

    Currents through calcium channels of members of an identified cluster of neurons (B cells) in the pond snail Lymnaea stagnalis were studied under voltage clamp. The normal physiological saline was modified to maximize the visibility of voltage-dependent calcium currents and minimize contamination by other currents. Barium was used as the charge carrier for the calcium channels. Depolarizing voltage steps induce an inward current, the magnitude of which varies with the barium concentration. In brains taken from animals not exposed in vivo to lead, in vitro addition of lead acetate to the recording medium (0.25 to 14 microM) inhibits the barium current by 59 +/- 14% (mean +/- s.d.), in a manner that is independent of the lead concentration. The magnitude of the residual current still varies with the barium concentration. The voltage dependence of the current appears to be unaffected by lead. In contrast to some other calcium-channel blockers, such as cobalt, the inhibition of barium currents by in vitro lead exposure is irreversible, at least in short-term experiments. Contrary to expectations based on these in vitro results, barium currents in B cells of animals exposed to 5 microM lead for 6 to 12 weeks in vivo were approximately twice as large as barium currents in B cells from unexposed controls, when both were recorded in lead-free saline. It is possible that chronic in vivo lead exposure causes an increase in the number of calcium channels in these neurons.

  10. Ropivacaine-Induced Contraction Is Attenuated by Both Endothelial Nitric Oxide and Voltage-Dependent Potassium Channels in Isolated Rat Aortae

    Directory of Open Access Journals (Sweden)

    Seong-Ho Ok

    2013-01-01

    Full Text Available This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, the neuronal NOS inhibitor Nω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP, the calcium-activated potassium channel inhibitor tetraethylammonium (TEA, the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, Nω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

  11. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca(2+) channels.

    Science.gov (United States)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin; Korotchenko, Svetlana; Lievens, Patricia Marie-Jeanne; Berezin, Vladimir; Dityatev, Alexander

    2015-12-01

    Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25 °C the binding of the peptide P7 (MGKMHKTCYN) was at ~50 μM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21 μM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23 μM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin-agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1.2 channels revealed that enzymatic digestion of highly sulfated heparan sulfates with heparinase 1 affects neither voltage-dependence of channel activation nor the level of steady state inactivation, but did speed up channel inactivation. Treatment of hippocampal cultures with heparinase 1 reduced the firing rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity.

  12. Altered calcium homeostasis in motor neurons following AMPA receptor but not voltage-dependent calcium channels' activation in a genetic model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Guatteo, Ezia; Carunchio, Irene; Pieri, Massimo; Albo, Federica; Canu, Nadia; Mercuri, Nicola B; Zona, Cristina

    2007-10-01

    Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brain stem and motor cortex. By combining electrophysiological recordings with imaging techniques, clearance/buffering capacity of cultured spinal cord motor neurons after a calcium accumulation has been analyzed in response to AMPA receptors' (AMPARs') activation and to depolarizing stimuli in a genetic mouse model of ALS (G93A). Our studies demonstrate that the amplitude of the calcium signal in response to AMPARs' or voltage-dependent calcium channels' activation is not significantly different in controls and G93A motor neurons. On the contrary, in G93A motor neurons, the [Ca(2+)](i) recovery to basal level is significantly slower compared to control neurons following AMPARs but not voltage-dependent calcium channels' activation. This difference was not observed in G93A cultured cortical neurons. This observation is the first to indicate a specific alteration of the calcium clearance linked to AMPA receptors' activation in G93A motor neurons and the involvement of AMPA receptor regulatory proteins controlling both AMPA receptor functionality and the sequence of events connected to them.

  13. Flow- and voltage-dependent blocking effect of ethosuximide on the inward rectifier K⁺ (Kir2.1) channel.

    Science.gov (United States)

    Huang, Chiung-Wei; Kuo, Chung-Chin

    2015-08-01

    Absence seizures are manifestations of abnormal thalamocortical oscillations characterized by spike-and-wave complexes in EEG. Ethosuximide (ETX) is one of the principal medications against absence seizures. We investigate the effect of ETX on the Kir2.1 channel, a prototypical inward rectifier K(+) channel possibly playing an important role in the setting of neuronal membrane potential. We demonstrate that the outward currents of Kir2.1 channels are significantly inhibited by intracellular ETX. We further show that the movement of neutral molecule ETX in the Kir2.1 channel is accompanied by ∼1.2 K(+), giving rise to the vivid voltage dependence of ETX unbinding rate. Moreover, the apparent affinity (K d ) of ETX in the channels are decreased by single-point mutations involving M183, E224, and S165, and especially by double mutations involving T141/S165, which always also disrupt the flux-coupling feature of ETX block. Molecular dynamics simulation demonstrates narrowing of the pore at ∼D172 by binding of ETX to S165 or T141. ETX block of the Kir2.1 channels may cause a modest but critical depolarization of the relevant neurons, decreasing available T-type Ca(2+) channels and consequently lessening pathological thalamocortical burst discharges.

  14. Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca²⁺ channels.

    Science.gov (United States)

    Takada, Yoshinori; Hirano, Mitsuru; Kiyonaka, Shigeki; Ueda, Yoshifumi; Yamaguchi, Kazuma; Nakahara, Keiko; Mori, Masayuki X; Mori, Yasuo

    2015-09-01

    Autism is a neurodevelopmental psychiatric disorder characterized by impaired reciprocal social interaction, disrupted communication, and restricted and stereotyped patterns of interests. Autism is known to have a strong genetic component. Although mutations in several genes account for only a small proportion of individuals with autism, they provide insight into potential biological mechanisms that underlie autism, such as dysfunction in Ca(2+) signaling, synaptic dysfunction, and abnormal brain connectivity. In autism patients, two mutations have been reported in the Rab3 interacting molecule 3 (RIM3) gene. We have previously demonstrated that RIM3 physically and functionally interacts with voltage-dependent Ca(2+) channels (VDCCs) expressed in neurons via the β subunits, and increases neurotransmitter release. Here, by introducing corresponding autism-associated mutations that replace glutamic acid residue 176 with alanine (E176A) and methionine residue 259 with valine (M259V) into the C2B domain of mouse RIM3, we demonstrate that both mutations partly cancel the suppressive RIM3 effect on voltage-dependent inactivation of Ba(2+) currents through P/Q-type CaV2.1 recombinantly expressed in HEK293 cells. In recombinant N-type CaV2.2 VDCCs, the attenuation of the suppressive RIM3 effect on voltage-dependent inactivation is conserved for M259V but not E176A. Slowing of activation speed of P/Q-type CaV2.1 currents by RIM3 is abolished in E176A, while the physical interaction between RIM3 and β subunits is significantly attenuated in M259V. Moreover, increases by RIM3 in depolarization-induced Ca(2+) influx and acetylcholine release are significantly attenuated by E176A in rat pheochromocytoma PC12 cells. Thus, our data raise the interesting possibility that autism phenotypes are elicited by synaptic dysfunction via altered regulation of presynaptic VDCC function and neurotransmitter release.

  15. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells.

    Science.gov (United States)

    Onetti, C G; Lara, J; García, E

    1996-05-01

    Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

  16. Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2 Is Involved in ABA-Mediated Early Seedling Development

    Directory of Open Access Journals (Sweden)

    Xufeng Li

    2009-05-01

    Full Text Available The voltage-dependent anion channel (VDAC is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana.

  17. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Science.gov (United States)

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

    2012-01-01

    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  18. Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

    Science.gov (United States)

    Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

    2012-01-01

    The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

  19. Expression and localization of voltage dependent potassium channel Kv4.2 in epilepsy associated focal lesions

    NARCIS (Netherlands)

    Aronica, E.; Boer, K.; Doorn, K.J.; Zurolo, E.; Spliet, W.G.M.; van Rijen, P.C.; Baayen, J.C.; Gorter, J.A.; Jeromin, A.

    2009-01-01

    An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal

  20. Expression and localization of voltage dependent potassium channel Kv4.2 in epilepsy associated focal lesions

    NARCIS (Netherlands)

    Aronica, E.; Boer, K.; Doorn, K.J.; Zurolo, E.; Spliet, W.G.M.; van Rijen, P.C.; Baayen, J.C.; Gorter, J.A.; Jeromin, A.

    2009-01-01

    An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal

  1. A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

    Science.gov (United States)

    Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

    2013-06-01

    The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

  2. Channel protein engineering: Synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers

    OpenAIRE

    1988-01-01

    A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nons...

  3. Aspartic Acid Residue D3 Critically Determines Cx50 Gap Junction Channel Transjunctional Voltage-Dependent Gating and Unitary Conductance

    Science.gov (United States)

    Xin, Li; Nakagawa, So; Tsukihara, Tomitake; Bai, Donglin

    2012-01-01

    Previous studies have suggested that the aspartic acid residue (D) at the third position is critical in determining the voltage polarity of fast Vj-gating of Cx50 channels. To test whether another negatively charged residue (a glutamic acid residue, E) could fulfill the role of the D3 residue, we generated the mutant Cx50D3E. Vj-dependent gating properties of this mutant channel were characterized by double-patch-clamp recordings in N2A cells. Macroscopically, the D3E substitution reduced the residual conductance (Gmin) to near zero and outwardly shifted the half-inactivation voltage (V0), which is a result of both a reduced aggregate gating charge (z) and a reduced free-energy difference between the open and closed states. Single Cx50D3E gap junction channels showed reduced unitary conductance (γj) of the main open state, reduced open dwell time at ±40 mV, and absence of a long-lived substate. In contrast, a G8E substitution tested to compare the effects of the E residue at the third and eighth positions did not modify the Vj-dependent gating profile or γj. In summary, this study is the first that we know of to suggest that the D3 residue plays an essential role, in addition to serving as a negative-charge provider, as a critical determinant of the Vj-dependent gating sensitivity, open-closed stability, and unitary conductance of Cx50 gap junction channels. PMID:22404924

  4. Voltage dependence of cardiac excitation-contraction coupling: unitary Ca2+ current amplitude and open channel probability.

    Science.gov (United States)

    Altamirano, Julio; Bers, Donald M

    2007-09-14

    Excitation-contraction coupling in cardiac myocytes occurs by Ca2+-induced Ca2+ release, where L-type Ca2+ current evokes a larger sarcoplasmic reticulum (SR) Ca2+ release. The Ca2+-induced Ca2+ release amplification factor or gain (SR Ca2+ release/I(Ca)) is usually assessed by the V(m) dependence of current and Ca2+ transients. Gain rises at negative V(m), as does single channel I(Ca) (i(Ca)), which has led to the suggestion that the increases of i(Ca) amplitude enhances gain at more negative V(m). However, I(Ca) = NP(o) x i(Ca) (where NP(o) is the number of open channels), and NP(o) and i(Ca) both depend on V(m). To assess how i(Ca) and NP(o) separately influence Ca2+-induced Ca2+ release, we measured I(Ca) and junctional SR Ca2+ release in voltage-clamped rat ventricular myocytes using "Ca2+ spikes" (confocal microscopy). To vary i(Ca) alone, we changed [Ca2+](o) rapidly at constant test V(m) (0 mV) or abruptly repolarized from +120 mV to different V(m) (at constant [Ca2+](o)). To vary NP(o) alone, we altered Ca2+ channel availability by varying holding V(m) (at constant test V(m)). Reducing either i(Ca) or NP(o) alone increased excitation-contraction coupling gain. Thus, increasing i(Ca) does not increase gain at progressively negative test V(m). Such enhanced gain depends on lower NP(o) and reduced redundant Ca2+ channel openings (per junction) and a consequently smaller denominator in the gain equation. Furthermore, modest i(Ca) (at V(m) = 0 mV) may still effectively trigger SR Ca2+ release, whereas at positive V(m) (and smaller i(Ca)), high and well-synchronized channel openings are required for efficient excitation-contraction coupling. At very positive V(m), reduced i(Ca) must explain reduced SR Ca2+ release.

  5. α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease.

    Science.gov (United States)

    Rostovtseva, Tatiana K; Gurnev, Philip A; Protchenko, Olga; Hoogerheide, David P; Yap, Thai Leong; Philpott, Caroline C; Lee, Jennifer C; Bezrukov, Sergey M

    2015-07-24

    Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.

  6. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired β-adrenoceptor-mediated relaxation of renal arteries in hypertension.

    Science.gov (United States)

    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei; Davis, Alison J; Jepps, Thomas A; Olesen, Søren P; Cole, William C; Moffatt, James D; Greenwood, Iain A

    2012-04-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

  7. Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers.

    Science.gov (United States)

    Decher, Niels; Chen, Jun; Sanguinetti, Michael C

    2004-04-02

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.

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

    Science.gov (United States)

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

    2016-01-18

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

  9. A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis.

    Directory of Open Access Journals (Sweden)

    Jessica A Hennessey

    Full Text Available Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS, and Timothy syndrome (TS. We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680 predicting a glycine>arginine substitution at residue 1911 (p.G1911R of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID, for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of

  10. Lidocaine block of cardiac sodium channels.

    Science.gov (United States)

    Bean, B P; Cohen, C J; Tsien, R W

    1983-05-01

    Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 muM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 muM, at a negative holding potential where inactivation was completely removed, to approximately 10 muM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (tauless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (tau approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 muM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 muM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the "modulated-receptor hypothesis" of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.

  11. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: cihima@inje.ac.kr [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: parkws@kangwon.ac.kr [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)

    2015-06-15

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  12. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    Science.gov (United States)

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

  13. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin

    Science.gov (United States)

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; de Oliveira Alvares, Lucas

    2016-01-01

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca2+ channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca2+ influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time. PMID:26947131

  14. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin.

    Science.gov (United States)

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; Alvares, Lucas de Oliveira

    2016-03-07

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca(2+) channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca(2+) influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time.

  15. Differential rescue of spatial memory deficits in aged rats by L-type voltage-dependent calcium channel and ryanodine receptor antagonism.

    Science.gov (United States)

    Hopp, S C; D'Angelo, H M; Royer, S E; Kaercher, R M; Adzovic, L; Wenk, G L

    2014-11-01

    Age-associated memory impairments may result as a consequence of neuroinflammatory induction of intracellular calcium (Ca(+2)) dysregulation. Altered L-type voltage-dependent calcium channel (L-VDCC) and ryanodine receptor (RyR) activity may underlie age-associated learning and memory impairments. Various neuroinflammatory markers are associated with increased activity of both L-VDCCs and RyRs, and increased neuroinflammation is associated with normal aging. In vitro, pharmacological blockade of L-VDCCs and RyRs has been shown to be anti-inflammatory. Here, we examined whether pharmacological blockade of L-VDCCs or RyRs with the drugs nimodipine and dantrolene, respectively, could improve spatial memory and reduce age-associated increases in microglia activation. Dantrolene and nimodipine differentially attenuated age-associated spatial memory deficits but were not anti-inflammatory in vivo. Furthermore, RyR gene expression was inversely correlated with spatial memory, highlighting the central role of Ca(+2) dysregulation in age-associated memory deficits.

  16. Alterations of voltage-dependent calcium channel currents in basilar artery smooth muscle cells at early stage of subarachnoid hemorrhage in a rabbit model.

    Directory of Open Access Journals (Sweden)

    Xianqing Shi

    Full Text Available OBJECTIVE: To investigate the changes in the currents of voltage-dependent calcium channels (VDCCs in smooth muscle cells of basilar artery in a rabbit model of subarachnoid hemorrhage (SAH. METHODS: New Zealand white rabbits were randomly divided into five groups: sham (C, normal (N, 24 hours (S1, 48 hours (S2 and 72 hours (S3 after SAH. Non-heparinized autologous arterial blood (1 ml/kg was injected into the cisterna magna to create SAH after intravenous anesthesia, and 1 ml/kg of saline was injected into cisterna magna in the sham group. Rabbits in group N received no injections. Basilar artery in S1, S2, S3 group were isolated at 24, 48, 72 hours after SAH. Basilar artery in group C was isolated at 72 hours after physiological saline injection. Basilar artery smooth muscle cells were isolated for all groups. Whole-cell patch-clamp technique was utilized to record cell membrane capacitance and VDCCs currents. The VDCCs antagonist nifedipine was added to the bath solution to block the Ca(++ channels currents. RESULTS: There were no significant differences in the number of cells isolated, the cell size and membrane capacitance among all the five groups. VDCC currents in the S1-S3 groups had higher amplitudes than those in control and sham groups. The significant change of current amplitude was observed at 72 hours after SAH, which was higher than those of 24 and 48 hours. The VDCCs were shown to expression in human artery smooth muscle cells. CONCLUSIONS: The changes of activation characteristics and voltage-current relationship at 72 hours after SAH might be an important event which leads to a series of molecular events in the microenvironment of the basilar artery smooth muscle cells. This may be the key time point for potential therapeutic intervention against subarachnoid hemorrhage.

  17. 线粒体电压依赖性阴离子通道与心血管疾病%Voltage-dependent Anion Channel and Cardiovascular Diseases

    Institute of Scientific and Technical Information of China (English)

    夏晶

    2013-01-01

    电压依赖性阴离子通道(VDAC)是位于线粒体外膜的通道蛋白,是线粒体与细胞质之间转运ATP以及其他代谢产物的主要通道,在线粒体代谢和细胞生长中发挥重要调控作用.近期研究发现,在心肌缺血再灌、糖尿病、心衰、高血压和动脉粥样硬化时,VDAC表达明显增加,引起细胞内钙离子循环紊乱、氧化应激,进而导致细胞凋亡,已成为心血管疾病研究的新热点.本文就VDAC的分子功能,调控及其在心血管疾病中的作用和相关机制进行综述.%The voltage-dependent anion channel (VDAC),a mitochondrial membrane channel protein located in the outer of mitochondrial membrane,is the main pathway between mitochondria and cytoplasm exchanging ADP,ATP,and other metabolites,and plays an important role in mitochondrial metabolism and cell growth.A growing evidence showed that VDAC was increased in cardiovascular diseases including myocardial ischemia and reperfusion,diabetes,heart failure,hypertension and atherosclerosis.The abnormal state of VDAC will result in cell death by inducing calcium cycling dysfunction and oxidative stress.And VDAC has become a hot topic in the field of cardiovascular diseases research.In this article,we will introduce the molecular function and regulation of VDAC and its role in cardiovascular diseases.

  18. Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers

    Science.gov (United States)

    1987-01-01

    Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found. PMID:2443607

  19. Delta receptors are required for full inhibitory coupling of mu-receptors to voltage-dependent Ca(2+) channels in dorsal root ganglion neurons.

    Science.gov (United States)

    Walwyn, Wendy; John, Scott; Maga, Matthew; Evans, Christopher J; Hales, Tim G

    2009-07-01

    Recombinant micro and delta opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca(2+) channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined micro-receptor coupling to VDCCs in dorsal root ganglion neurons of delta(+/+), delta(+/-), and delta(-/-) mice. Neurons deficient in delta receptors exhibited reduced inhibition of VDCCs by morphine and [D-Ala(2),Phe(4),Gly(5)-ol]-enkephalin (DAMGO). An absence of delta receptors caused reduced efficacy of DAMGO without affecting potency. An absence of delta receptors reduced neither the density of VDCCs nor their inhibition by either the GABA(B) receptor agonist baclofen or intracellular guanosine 5'-O-(3-thio)triphosphate. Flow cytometry revealed a reduction in micro-receptor surface expression in delta(-/-) neurons without altered DAMGO-induced internalization. There was no change in micro-receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)-sensitive mu-receptor-coupling efficacy was fully restored to delta(+/+) levels in delta(-/-) neurons by expression of recombinant delta receptors. However, the dimerization-deficient delta-15 construct expressed in delta(-/-) neurons failed to fully restore the inhibitory coupling of micro-receptors compared with that seen in delta(+/+) neurons, suggesting that, although not essential for micro-receptor function, micro-delta receptor dimerization contributes to full micro-agonist efficacy. Because DAMGO exhibited a similar potency in delta(+/+) and delta(-/-) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.

  20. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Science.gov (United States)

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  1. Voltage-dependent anion channels (VDACs) promote mitophagy to protect neuron from death in an early brain injury following a subarachnoid hemorrhage in rats.

    Science.gov (United States)

    Li, Jian; Lu, Jianfei; Mi, Yongjie; Shi, Zhao; Chen, Chunhua; Riley, John; Zhou, Changman

    2014-07-21

    The term mitophagy is coined to describe the selective removal of mitochondria by autophagy but the process itself is still contentious, especially in the early period following subarachnoid hemorrhage (SAH). In the present study, we investigated the role of mitophagy following 48h after SAH injury in rats. Specifically evaluating whether mitophagy, through voltage dependant anion channels (VDACs) interacting with microtubule-associated protein 1 light chain 3, could orchestrate the induction of apoptotic and necrotic cell death in neurons, a VDAC1siRNA and an activitor Rapamycian (RAPA), were engaged. One hundred and twelve male Sprague-Dawley rats were randomly divided into 4 groups: Sham, SAH, SAH+VDAC1siRNA, and SAH+RAPA. Outcomes measured included mortality rate, brain edema, BBB disruption, and neurobehavioral testing. We also used western blotting techniques to analyze the expressions of key mitophagic/autophagic proteins and pro-apoptotic protein such as ROS, VDAC1, LC-3II and Caspase-3. Rapamycin treatment significantly improved the mortality rate, cerebral edema, and neurobehavioral deficits; apoptotic and necrotic cell death in neurons were reduced by Rapamycin following SAH injury. However, VDAC1siRNA worsened the brain injury following SAH. Immunohistochemical staining and western blot analysis demonstrated a decreased expression of VDAC1, LC3II, and an increase of ROS and Caspase-3 followed by VDAC1siRNA administration. In conclusion, mitophagy induced by VDAC1 following SAH injury may in fact play a significant role in neuroprotection, the mechanism which may be through the attenuation of the apoptosic and necrosic molecular pathways. This translates a preservation of functional integrity and an improvement in mortality.

  2. Transcriptional upregulation of α2δ-1 elevates arterial smooth muscle cell voltage-dependent Ca2+ channel surface expression and cerebrovascular constriction in genetic hypertension.

    Science.gov (United States)

    Bannister, John P; Bulley, Simon; Narayanan, Damodaran; Thomas-Gatewood, Candice; Luzny, Patrik; Pachuau, Judith; Jaggar, Jonathan H

    2012-10-01

    A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1:CaV1.2α1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

  3. Action of the pyrethroid insecticide cypermethrin on rat brain IIa sodium channels expressed in xenopus oocytes.

    Science.gov (United States)

    Smith, T J; Soderlund, D M

    1998-12-01

    Pyrethroid insecticides bind to a unique site on voltage-dependent sodium channels and prolong sodium currents, leading to repetitive bursts of action potentials or use-dependent nerve block. To further characterize the site and mode of action of pyrethroids on sodium channels, we injected synthetic mRNA encoding the rat brain IIa sodium channel alpha subunit, either alone or in combination with synthetic mRNA encoding the rat sodium channel beta1 subunit, into oocytes of the frog Xenopus laevis and assessed the actions of the pyrethroid insecticide [1R,cis,alphaS]-cypermethrin on expressed sodium currents by two-electrode voltage clamp. In oocytes expressing only the rat brain IIa alpha subunit, cypermethrin produced a slowly-decaying sodium tail current following a depolarizing pulse. In parallel experiments using oocytes expressing the rat brain IIa alpha subunit in combination with the rat beta1 subunit, cypermethrin produced qualitatively similar tail currents following a depolarizing pulse and also induced a sustained component of the sodium current measured during a step depolarization of the oocyte membrane. The voltage dependence of activation and steady-state inactivation of the cypermethrin-dependent sustained current were identical to those of the peak transient sodium current measured in the absence of cypermethrin. Concentration-response curves obtained using normalized tail current amplitude as an index of the extent of sodium channel modification by cypermethrin revealed that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit increased the apparent affinity of the sodium channel binding site for cypermethrin by more than 20-fold. These results confirm that the pyrethroid binding site is intrinsic to the sodium channel alpha subunit and demonstrate that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit alters the apparent affinity of this site for pyrethroids.

  4. Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor.

    Science.gov (United States)

    Parkash, Jai

    2008-08-01

    The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues

  5. Modification of sodium and potassium channel kinetics by diethyl ether and studies on sodium channel inactivation in the crayfish giant axon membrane

    Energy Technology Data Exchange (ETDEWEB)

    Bean, Bruce Palmer

    1979-01-01

    The effects of ether and halothane on membrane currents in the voltage clamped crayfish giant axon membrane were investigated. Concentrations of ether up to 300 mM and of halothane up to 32 mM had no effect on resting potential or leakage conductance. Ether and halothane reduced the size of sodium currents without changing the voltage dependence of the peak currents or their reversal potential. Ether and halothane also produced a reversible, dose-dependent speeding of sodium current decay at all membrane potentials. Ether reduced the time constants for inactivation, and also shifted the midpoint of the steady-state inactivation curve in the hyperpolarizing direction. Potassium currents were smaller with ether present, with no change in the voltage dependence of steady-state currents. The activation of potassium channels was faster with ether present. There was no apparent change in the capacitance of the crayfish giant axon membrane with ether concentrations of up to 100 mM. Experiments on sodium channel inactivation kinetics were performed using 4-aminopyridine to block potassium currents. Sodium currents decayed with a time course generally fit well by a single exponential. The time constant of decay was a steep function of voltage, especially in the negative resistance region of the peak current vs voltage relation.The time course of inactivation was very similar to that of the decay of the current at the same potential. The measurement of steady-state inactivation curves with different test pulses showed no shifts along the voltage asix. The voltage-dependence of the integral of sodium conductance was measured to test models of sodium channel inactivation in which channels must open before inactivating; the results appear inconsistent with some of the simplest cases of such models.

  6. Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Sara Dizayee

    Full Text Available BACKGROUND: Two pertussis toxin sensitive G(i proteins, G(i2 and G(i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i isoforms are functionally distinct. To test for isoform-specific functions of G(i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC. METHODS: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2 (Gα(i2 (-/- or Gα(i3 (Gα(i3 (-/-. mRNA levels of Gα(i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i and Ca(vα(1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2 phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. RESULTS: In cardiac tissue from Gα(i2 (-/- mice, Gα(i3 mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3 (-/- mouse hearts, Gα(i2 mRNA (127 ± 5% and protein (131 ± 10% levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2 (-/- mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05 compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22, whereas it was increased in myocytes from Gα(i3 (-/- mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05. Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2 (but not of Gα(i3 and following treatment with pertussis toxin in Gα(i3 (-/-. The pore forming Ca(vα(1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(vα(1 and Ca(vβ(2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2. CONCLUSION: Our data provide novel evidence for an isoform

  7. Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating.

    Science.gov (United States)

    Yau, Hau-Jie; Baranauskas, Gytis; Martina, Marco

    2010-10-15

    The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

  8. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Birn, Pia; Hansen, Anker J

    2004-01-01

    be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100...

  9. Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons

    Directory of Open Access Journals (Sweden)

    Smith Paula L

    2011-09-01

    Full Text Available Abstract Background Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. Results Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. Conclusions Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

  10. EFFECTS OF GLUTAMATE ON SODIUM CHANNEL IN ACUTELY DISSOCIATED HIPPOCAMPAL CA1 PYRAMIDAL NEURONS OF RATS

    Institute of Scientific and Technical Information of China (English)

    高宾丽; 伍国锋; 杨艳; 刘智飞; 曾晓荣

    2011-01-01

    Objective To observe the effects of glutamate on sodium channel in acutely dissociated hippocampal CA1 pyramidal neurons of rats.Methods Voltage-dependent sodium currents (INa) in acutely dissociated hippocampal CA1 pyramidal neurons of neonate rats were recorded by whole-cell patchclamp of the brain slice technique when a series of doses of glutamate (100-1000μmol/L) were applied.Results Different concentrations of glutamate could inhibit INa,and higher concentration of glutamate affected greater inhibitio...

  11. Chronic electroconvulsive stimulation but not chronic restraint stress modulates mRNA expression of voltage-dependent potassium channels Kv7.2 and Kv11.1 in the rat piriform cortex

    DEFF Research Database (Denmark)

    Hjæresen, Marie-Louise; Hageman, Ida; Plenge, Per

    2008-01-01

    The mechanisms by which stress and electroconvulsive therapy exert opposite effects on the course of major depression are not known. Potential candidates might include the voltage-dependent potassium channels. Potassium channels play an important role in maintaining the resting membrane potential...... and controlling neuronal excitability. To explore this hypothesis, we examined the effects of one or several electroconvulsive stimulations and chronic restraint stress (6 h/day for 21 days) on the expression of voltage-dependent potassium channel Kv7.2, Kv11.1, and Kv11.3 mRNA in the rat brain using in situ...... hybridization. Repeated, but not acute, electroconvulsive stimulation increased Kv7.2 and Kv11.1 mRNA levels in the piriform cortex. In contrast, restraint stress had no significant effect on mRNA expression of Kv7.2, Kv11.1, or Kv11.3 in any of the brain regions examined. Thus, it appears that the investigated...

  12. JZTX-IV, a unique acidic sodium channel toxin isolated from the spider Chilobrachys jingzhao.

    Science.gov (United States)

    Wang, Meichi; Diao, Jianbo; Li, Jiang; Tang, Jianzhou; Lin, Yin; Hu, Weijun; Zhang, Yongqun; Xiao, Yucheng; Liang, Songping

    2008-12-15

    Neurotoxins are important tools to explore the structure and function relationship of different ion channels. From the venom of Chinese spider Chilobrachys jingzhao, a novel toxin, Jingzhaotoxin-IV (JZTX-IV), is isolated and characterized. It consists of 34 amino acid residues including six acidic residues clustered with negative charge (pI=4.29). The full-length cDNA of JZTX-IV encodes an 86-amino acid precursor containing a signal peptide of 21 residues, a mature peptide of 34 residues and an intervening sequence of 29 residues with terminal Lys-Gly as the signal of amidation. Under whole-cell patch clamp conditions, JZTX-IV inhibits current and slows the inactivation of sodium channels by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. In addition, JZTX-IV shows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons, differs from the classic site 3 toxins that do not affect the steady-state of inactivation. At high concentration, JZTX-IV has no significant effect on tetrodotoxin-resistant (TTX-R) sodium channels on rat DRG neurons and tetrodotoxin-sensitive (TTX-S) sodium channels on hippocampal neurons. Our data establish that, contrary to known toxins, JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating, thus making it a novel probe of channel gating in sodium channels with potential to shed new light on this process.

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

    Directory of Open Access Journals (Sweden)

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

    2011-01-01

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

  14. Docking Studies of Phthalimide Pharmacophore as a Sodium Channel Blocker

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    Maryam Iman

    2013-09-01

    Full Text Available   Objective(s: Recently, phthalimide derivatives were designed based on ameltolide and thalidomide as they possess a similar degree of anticonvulsant potency due to their phenytoin-like profile. The ability of phthalimide pharmacophore to interact with neuronal voltage-dependent sodium channels was studied in the batrachotoxin affinity assay. Therefore, in the present study, a series of 19 compounds of phthalimide pharmacophore possessing a variety of substituents (NO2, NH2 , Me, Cl, COOH, MeO at 2-, 3-, and 4- position of the N-phenyl ring and N-(3-amino-2-methylphenyl succinimide, were subjected to docking studies in order to inhibit voltage-gated sodium channels.   Materials and Methods : Chemical structures of all compounds were designed using HYPERCHEM program and Conformational studies were performed through semi-empirical molecular orbital calculations method followed by PM3 force field. Total energy gradient calculated as a root mean square (RMS value, until the RMS gradient was 0.01 kcal mol-1. Among all energy minima conformers, the global minimum of compounds was used in docking calculations. Using a model of the open pore of Na channels, docking study was performed by AUTODOCK4.2 program. Results : Docking studies have revealed that these types of ligands interacted mainly with II-S6 residues of NaV1.2 through making hydrogen bonds and have additional hydrophobic interactions with domain I, II, III and IV in the channel's inner pore. Conclusion   : These computational studies have displayed that these compounds are capable of inhibiting Na channel, efficiently.

  15. Morphine decreases enteric neuron excitability via inhibition of sodium channels.

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    Tricia H Smith

    Full Text Available Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP. Cells with AHP expressed greater density of sodium currents. Morphine (3 µM significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours, action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.

  16. Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

    Science.gov (United States)

    Morera, Francisco J; Saravia, Julia; Pontigo, Juan Pablo; Vargas-Chacoff, Luis; Contreras, Gustavo F; Pupo, Amaury; Lorenzo, Yenisleidy; Castillo, Karen; Tilegenova, Cholpon; Cuello, Luis G; Gonzalez, Carlos

    2015-11-01

    Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Brain Sodium Channel and Febrile Seizure Mechanism

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2009-10-01

    Full Text Available Researchers at the University of Melbourne, Australia, measured the effect of temperature on brain sodium channel, Nav1.2, properties, using a computer model of the dentate gyrus granule cell.

  18. Neuronal Sodium Channels in Neurodegeneration and Neuroprotection

    Science.gov (United States)

    2002-06-01

    NIL) of the Univ Michigan. Ann Arbor Mr, USA. rat pituitary gland exhibit a 40-50% decrease in sodium current density from postnatal day 3 (P3) to...that deleted all residues beyond the tyr (gIL182STOP). Ankyrin rec of channel production may underlie the suppression of sodium current density. To...subunits con- 11 critical is for ankyrin association. Homophilic cell adhesion through 81 subunits m tributes to the suppression of melanotrope sodium

  19. Sodium channel biophysics, late sodium current and genetic arrhythmic syndromes.

    Science.gov (United States)

    Chadda, Karan R; Jeevaratnam, Kamalan; Lei, Ming; Huang, Christopher L-H

    2017-03-06

    Arrhythmias arise from breakdown of orderly action potential (AP) activation, propagation and recovery driven by interactive opening and closing of successive voltage-gated ion channels, in which one or more Na(+) current components play critical parts. Early peak, Na(+) currents (I Na) reflecting channel activation drive the AP upstroke central to cellular activation and its propagation. Sustained late Na(+) currents (I Na-L) include contributions from a component with a delayed inactivation timecourse influencing AP duration (APD) and refractoriness, potentially causing pro-arrhythmic phenotypes. The magnitude of I Na-L can be analysed through overlaps or otherwise in the overall voltage dependences of the steady-state properties and kinetics of activation and inactivation of the Na(+) conductance. This was useful in analysing repetitive firing associated with paramyotonia congenita in skeletal muscle. Similarly, genetic cardiac Na(+) channel abnormalities increasing I Na-L are implicated in triggering phenomena of automaticity, early and delayed afterdepolarisations and arrhythmic substrate. This review illustrates a wide range of situations that may accentuate I Na-L. These include (1) overlaps between steady-state activation and inactivation increasing window current, (2) kinetic deficiencies in Na(+) channel inactivation leading to bursting phenomena associated with repetitive channel openings and (3) non-equilibrium gating processes causing channel re-opening due to more rapid recoveries from inactivation. All these biophysical possibilities were identified in a selection of abnormal human SCN5A genotypes. The latter presented as a broad range of clinical arrhythmic phenotypes, for which effective therapeutic intervention would require specific identification and targeting of the diverse electrophysiological abnormalities underlying their increased I Na-L.

  20. Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

    Science.gov (United States)

    Kulke, Daniel; von Samson-Himmelstjerna, Georg; Miltsch, Sandra M; Wolstenholme, Adrian J; Jex, Aaron R; Gasser, Robin B; Ballesteros, Cristina; Geary, Timothy G; Keiser, Jennifer; Townson, Simon; Harder, Achim; Krücken, Jürgen

    2014-12-01

    The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among

  1. Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

    Directory of Open Access Journals (Sweden)

    Daniel Kulke

    2014-12-01

    Full Text Available The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in

  2. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

    Science.gov (United States)

    Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K; Craik, David J; Kent, Stephen B H; French, Robert J; Bezanilla, Francisco; Correa, Ana M

    2017-03-07

    Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

  3. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

    Science.gov (United States)

    Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K.; Craik, David J.; Kent, Stephen B. H.; French, Robert J.; Bezanilla, Francisco; Correa, Ana M.

    2017-01-01

    Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating. PMID:28202723

  4. Potentiation of prolactin secretion following lactotrope escape from dopamine action. II. Phosphorylation of the alpha(1) subunit of L-type, voltage-dependent calcium channels.

    Science.gov (United States)

    Hernández, M E; del Mar Hernández, M; Díaz-Muñoz, M; Clapp, C; de la Escalera, G M

    1999-07-01

    Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2

  5. Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin.

    Science.gov (United States)

    Cestèle, Sandrine; Yarov-Yarovoy, Vladimir; Qu, Yusheng; Sampieri, François; Scheuer, Todd; Catterall, William A

    2006-07-28

    Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

  6. Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin*S

    Science.gov (United States)

    Cestèle, Sandrine; Yarov-Yarovoy, Vladimir; Qu, Yusheng; Sampieri, François; Scheuer, Todd; Catterall, William A.

    2006-01-01

    Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. β-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that β-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu779 in extracellular loop IIS1–S2 and both Glu837 and Leu840 in extracellular loop IIS3–S4 reduce the binding affinity of β-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect β-scorpion toxin binding but alter voltage dependence of activation and enhance β-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states. PMID:16679310

  7. Analysis of the action of lidocaine on insect sodium channels

    OpenAIRE

    Song, Weizhong; Silver, Kristopher S.; Du, Yuzhe; Liu, Zhiqi; Dong, Ke

    2010-01-01

    A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are n...

  8. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    M), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombinant as well as native full-length neuronal Cav1.2α1 channels was confirmed using the heparin–agarose pull down assay. Whole cell patch clamp recordings in HEK293 cells transfected with neuronal Cav1...... rate and led to appearance of long-lasting bursts in the same manner as treatment with the inhibitor of L-VDCC diltiazem. Thus, heparan sulfate proteoglycans may bind to and regulate L-VDCC inactivation and network activity....

  9. Divergent sodium channel defects in familial hemiplegic migraine.

    Science.gov (United States)

    Kahlig, Kristopher M; Rhodes, Thomas H; Pusch, Michael; Freilinger, Tobias; Pereira-Monteiro, José M; Ferrari, Michel D; van den Maagdenberg, Arn M J M; Dichgans, Martin; George, Alfred L

    2008-07-15

    Familial hemiplegic migraine type 3 (FHM3) is a severe autosomal dominant migraine disorder caused by mutations in the voltage-gated sodium channel Na(V)1.1 encoded by SCN1A. We determined the functional consequences of three mutations linked to FHM3 (L263V, Q1489K, and L1649Q) in an effort to identify molecular defects that underlie this inherited migraine disorder. Only L263V and Q1489K generated quantifiable sodium currents when coexpressed in tsA201 cells with the human beta(1) and beta(2) accessory subunits. The third mutant, L1649Q, failed to generate measurable whole-cell current because of markedly reduced cell surface expression. Compared to WT-Na(V)1.1, Q1489K exhibited increased persistent current but also enhanced entry into slow inactivation as well as delayed recovery from fast and slow inactivation, thus resulting in a predominantly loss-of-function phenotype further demonstrated by a greater loss of channel availability during repetitive stimulation. In contrast, L263V exhibited gain-of-function features, including delayed entry into, as well as accelerated recovery from, fast inactivation; depolarizing shifts in the steady-state voltage dependence of fast and slow inactivation; increased persistent current; and delayed entry into slow inactivation. Notably, the two mutations (Q1489K and L1649Q) that exhibited partial or complete loss of function are linked to typical FHM, whereas the gain-of-function mutation L263V occurred in a family having both FHM and a high incidence of generalized epilepsy. We infer from these data that a complex spectrum of Na(V)1.1 defects can cause FHM3. Our results also emphasize the complex relationship between migraine and epilepsy and provide further evidence that both disorders may share common molecular mechanisms.

  10. Photoaffinity labeling of alpha- and beta- scorpion toxin receptors associated with rat brain sodium channel.

    Science.gov (United States)

    Darbon, H; Jover, E; Couraud, F; Rochat, H

    1983-09-15

    Azido nitrophenylaminoacetyl [125I]iodo derivative of toxin II from Centruroides suffusus suffusus, a beta-toxin, and azido nitrophenylaminoacetyl [125I]iodo derivative of toxin V from Leiurus quinquestriatus quinquestriatus, an alpha-toxin, have been covalently linked after binding to their receptor sites that are related to the voltage sensitive sodium channel present in rat brain synaptosomes. Both derivatives labeled two polypeptides of 253000 +/- 20000 and 35000 +/- 2000 mol. wt. Labeling was blocked for each derivative by a large excess of the corresponding native toxin but no cross inhibition was obtained. These results suggest that both alpha - and beta - scorpion toxin receptors are located on or near the same two membrane polypeptides which may be part of the voltage dependent sodium channel.

  11. Modulation of neuronal sodium channels by the sea anemone peptide BDS-I.

    Science.gov (United States)

    Liu, Pin; Jo, Sooyeon; Bean, Bruce P

    2012-06-01

    Blood-depressing substance I (BDS-I), a 43 amino-acid peptide from sea anemone venom, is used as a specific inhibitor of Kv3-family potassium channels. We found that BDS-I acts with even higher potency to modulate specific types of voltage-dependent sodium channels. In rat dorsal root ganglion (DRG) neurons, 3 μM BDS-I strongly enhanced tetrodotoxin (TTX)-sensitive sodium current but weakly inhibited TTX-resistant sodium current. In rat superior cervical ganglion (SCG) neurons, which express only TTX-sensitive sodium current, BDS-I enhanced current elicited by small depolarizations and slowed decay of currents at all voltages (EC(50) ∼ 300 nM). BDS-I acted with exceptionally high potency and efficacy on cloned human Nav1.7 channels, slowing inactivation by 6-fold, with an EC(50) of approximately 3 nM. BDS-I also slowed inactivation of sodium currents in N1E-115 neuroblastoma cells (mainly from Nav1.3 channels), with an EC(50) ∼ 600 nM. In hippocampal CA3 pyramidal neurons (mouse) and cerebellar Purkinje neurons (mouse and rat), BDS-I had only small effects on current decay (slowing inactivation by 20-50%), suggesting relatively weak sensitivity of Nav1.1 and Nav1.6 channels. The biggest effect of BDS-I in central neurons was to enhance resurgent current in Purkinje neurons, an effect reflected in enhancement of sodium current during the repolarization phase of Purkinje neuron action potentials. Overall, these results show that BDS-I acts to modulate sodium channel gating in a manner similar to previously known neurotoxin receptor site 3 anemone toxins but with different isoform sensitivity. Most notably, BDS-I acts with very high potency on human Nav1.7 channels.

  12. Epithelial Sodium and Chloride Channels and Asthma

    Institute of Scientific and Technical Information of China (English)

    Wen Wang; Hong-Long Ji

    2015-01-01

    Objective:To focus on the asthmatic pathogenesis and clinical manifestations related to epithelial sodium channel (ENaC)/chlorine ion channel.Data Sources:The data analyzed in this review were the English articles from 1980 to 2015 from journal databases,primarily PubMed and Google Scholar.The terms used in the literature search were:(1) ENaCs;cystic fibrosis (CF) transmembrane conductance regulator (CFTR);asthma/asthmatic,(2) ENaC/sodium salt;CF;asthma/asthmatic,(3) CFTR/chlorine ion channels;asthma/asthmatic,(4) ENaC/sodium channel/scnn1a/scnn1b/scnn1g/scnn1d/amiloride-sensitive/amiloride-inhibtable sodium channels/sodium salt;asthma/asthmatic,lung/pulmonary/respiratory/tracheal/alveolar,and (5) CFTR;CF;asthma/asthmatic (ti).Study Selection:These studies included randomized controlled trials or studies covering asthma pathogenesis and clinical manifestations related to ENaC/chlorine ion channels within the last 25 years (from 1990 to 2015).The data involving chronic obstructive pulmonary disease and CF obtained from individual studies were also reviewed by the authors.Results:Airway surface liquid dehydration can cause airway inflammation and obstruction.ENaC and CFTR are closely related to the airway mucociliary clearance.Ion transporters may play a critical role in pathogenesis of asthmatic exacerbations.Conclusions:Ion channels have been the center of many studies aiming to understand asthmatic pathophysiological mechanisms or to identify therapeutic targets for better control of the disease.

  13. State-dependent block of rat Nav1.4 sodium channels expressed in xenopus oocytes by pyrazoline-type insecticides.

    Science.gov (United States)

    Silver, Kristopher; Soderlund, David M

    2005-06-01

    Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Na(v)1.4 sodium channels at a membrane potential of -120 mV, but depolarization to -60 mV or -30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.

  14. Genotypic to expression profiling of bovine calcium channel, voltage-dependent, alpha-2/delta subunit 1 gene, and their association with bovine mastitis among Frieswal (HFX Sahiwal) crossbred cattle of Indian origin.

    Science.gov (United States)

    Deb, Rajib; Singh, Umesh; Kumar, Sushil; Kumar, Arun; Singh, Rani; Sengar, Gyanendra; Mann, Sandeep; Sharma, Arjava

    2014-04-01

    Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A>G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n=101) was comparatively higher than AG (35, n=70) and GG (14.5, n=29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

  15. Selective alteration of sodium channel gating by Australian funnel-web spider toxins.

    Science.gov (United States)

    Nicholson, G M; Little, M J; Tyler, M; Narahashi, T

    1996-01-01

    The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.

  16. Voltage Dependence of Supercapacitor Capacitance

    Directory of Open Access Journals (Sweden)

    Szewczyk Arkadiusz

    2016-09-01

    Full Text Available Electronic Double-Layer Capacitors (EDLC, called Supercapacitors (SC, are electronic devices that are capable to store a relatively high amount of energy in a small volume comparing to other types of capacitors. They are composed of an activated carbon layer and electrolyte solution. The charge is stored on electrodes, forming the Helmholtz layer, and in electrolyte. The capacitance of supercapacitor is voltage- dependent. We propose an experimental method, based on monitoring of charging and discharging a supercapacitor, which enables to evaluate the charge in an SC structure as well as the Capacitance-Voltage (C-V dependence. The measurement setup, method and experimental results of charging/discharging commercially available supercapacitors in various voltage and current conditions are presented. The total charge stored in an SC structure is proportional to the square of voltage at SC electrodes while the charge on electrodes increases linearly with the voltage on SC electrodes. The Helmholtz capacitance increases linearly with the voltage bias while a sublinear increase of total capacitance was found. The voltage on SC increases after the discharge of electrodes due to diffusion of charges from the electrolyte to the electrodes. We have found that the recovery voltage value is linearly proportional to the initial bias voltage value.

  17. Analysis of the action of lidocaine on insect sodium channels.

    Science.gov (United States)

    Song, Weizhong; Silver, Kristopher S; Du, Yuzhe; Liu, Zhiqi; Dong, Ke

    2011-01-01

    A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are not important for the action of SCBIs on insect sodium channels, suggesting unique interactions of SCBIs with insect sodium channels. However, the mechanism of action of LAs on insect sodium channels has not been investigated. In this study, we examined the effects of lidocaine on a cockroach sodium channel variant, BgNa(v)1-1a, and determined whether F1817 and Y1824 are also critical for the action of LAs on insect sodium channels. Lidocaine blocked BgNa(v)1-1a channels in the resting state with potency similar to that observed in mammalian sodium channels. Lidocaine also stabilized both fast-inactivated and slow-inactivated states of BgNa(v)1-1a channels, and caused a limited degree of use- and frequency-dependent block, major characteristics of LA action on mammalian sodium channels. Alanine substitutions of F1817 and Y1824 reduced the sensitivity of the BgNa(v)1-1a channel to the use-dependent block by lidocaine, but not to tonic blocking and inactivation stabilizing effects of lidocaine. Thus, similar to those on mammalian sodium channels, F1817 and Y1824 are important for the action of lidocaine on cockroach sodium channels. Our results suggest that the receptor sites for lidocaine and SCBIs are different on insect sodium channels.

  18. The Voltage-Dependent Anion Channel 1 (AtVDAC1 Negatively Regulates Plant Cold Responses during Germination and Seedling Development in Arabidopsis and Interacts with Calcium Sensor CBL1

    Directory of Open Access Journals (Sweden)

    Zhi-Yong Li

    2013-01-01

    Full Text Available The voltage-dependent anion channel (VDAC, a highly conserved major mitochondrial outer membrane protein, plays crucial roles in energy metabolism and metabolite transport. However, knowledge about the roles of the VDAC family in plants is limited. In this study, we investigated the expression pattern of VDAC1 in Arabidopsis and found that cold stress promoted the accumulation of VDAC1 transcripts in imbibed seeds and mature plants. Overexpression of VDAC1 reduced tolerance to cold stress in Arabidopsis. Phenotype analysis of VDAC1 T-DNA insertion mutant plants indicated that a vdac1 mutant line had faster germination kinetics under cold treatment and showed enhanced tolerance to freezing. The yeast two-hybrid system revealed that VDAC1 interacts with CBL1, a calcium sensor in plants. Like the vdac1, a cbl1 mutant also exhibited a higher seed germination rate. We conclude that both VDAC1 and CBL1 regulate cold stress responses during seed germination and plant development.

  19. Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels.

    Science.gov (United States)

    Shimomura, Takushi; Irie, Katsumasa; Nagura, Hitoshi; Imai, Tomoya; Fujiyoshi, Yoshinori

    2011-03-04

    Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.

  20. A global defect in scaling relationship between electrical activity and availability of muscle sodium channels in hyperkalemic periodic paralysis.

    Science.gov (United States)

    Melamed-Frank, M; Marom, S

    1999-07-01

    Hyperkalemic periodic paralysis (HyperPP) is a hereditary disorder characterized by alternate episodic attacks of muscle weakness and muscle myotonia. The most common mutation associated with HyperPP is a T704M substitution in the skeletal-muscle sodium channel. This mutation increases sodium persistent currents, alters voltage dependence of activation and impairs slow inactivation. The present study shows experimental evidence in support of a potentially important global defect caused by the T704M mutation. While the effective rate of recovery from slow inactivation, in both normal and mutated channels, is related to the duration of past activity by a power law function, the scaling power of the mutated channel is significantly greater. This difference between the channels offers a clue for an explanation to the wide range of time scales, history dependence, and the mixed myotonic/paralysis effect, which mark the clinical picture of HyperPP.

  1. Effects of the β1 auxiliary subunit on modification of Rat Na(v)1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin.

    Science.gov (United States)

    He, Bingjun; Soderlund, David M

    2016-01-15

    We expressed rat Nav1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Nav1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~18 mV for tefluthrin and ~24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~10-14 mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Nav1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons.

  2. Effects of the β1 Auxiliary Subunit on Modification of Rat Nav1.6 Sodium Channels Expressed in HEK293 Cells by the Pyrethroid Insecticides Tefluthrin and Deltamethrin

    Science.gov (United States)

    He, Bingjun; Soderlund, David M.

    2015-01-01

    We expressed rat Nav1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Nav1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~18 mV for tefluthrin and ~24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~10–14 mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Nav1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons. PMID:26708501

  3. Modification of sodium and potassium channel gating kinetics by ether and halothane

    Energy Technology Data Exchange (ETDEWEB)

    Bean, B.P.; Shrager, P.; Goldstein, D.A.

    1981-03-01

    The effects of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the rising phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rats of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out.

  4. Molecular biology of insect sodium channels and pyrethroid resistance.

    Science.gov (United States)

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S

    2014-07-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors.

  5. Molecular Biology of Insect Sodium Channels and Pyrethroid Resistance

    Science.gov (United States)

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S.

    2015-01-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Most of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. PMID:24704279

  6. Splice variants of Na(V1.7 sodium channels have distinct β subunit-dependent biophysical properties.

    Directory of Open Access Journals (Sweden)

    Clare Farmer

    Full Text Available Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027, the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012. The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

  7. On conduction in a bacterial sodium channel.

    Directory of Open Access Journals (Sweden)

    Simone Furini

    Full Text Available Voltage-gated Na⁺-channels are transmembrane proteins that are responsible for the fast depolarizing phase of the action potential in nerve and muscular cells. Selective permeability of Na⁺ over Ca²⁺ or K⁺ ions is essential for the biological function of Na⁺-channels. After the emergence of the first high-resolution structure of a Na⁺-channel, an anionic coordination site was proposed to confer Na⁺ selectivity through partial dehydration of Na⁺ via its direct interaction with conserved glutamate side chains. By combining molecular dynamics simulations and free-energy calculations, a low-energy permeation pathway for Na⁺ ion translocation through the selectivity filter of the recently determined crystal structure of a prokaryotic sodium channel from Arcobacter butzleri is characterised. The picture that emerges is that of a pore preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers. In contrast to K⁺-channels, the movements of the ions appear to be weakly coupled in Na⁺-channels. When the free-energy maps for Na⁺ and K⁺ ions are compared, a selective site is characterised in the narrowest region of the filter, where a hydrated Na⁺ ion, and not a hydrated K⁺ ion, is energetically stable.

  8. Statistical analysis of single sodium channels. Effects of N-bromoacetamide.

    Science.gov (United States)

    Horn, R; Vandenberg, C A; Lange, K

    1984-01-01

    Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with

  9. Kinetic changes and modulation by carbamazepine on voltage-gated sodium channels in rat CA1 neurons after epilepsy

    Institute of Scientific and Technical Information of China (English)

    Guang-chun SUN; Taco WERKMAN; Wytse J WADMAN

    2006-01-01

    Aim: To study whether the functional properties of sodium channels, and subsequently the channel modulation by carbamazepine (CBZ) in hippocampal CA1 neurons can be changed after epileptic seizures. Methods: We used the acutely dissociated hippocampal CA1 pyramidal cells from epilepsy model rats 3 weeks and 3 months respectively after kainate injection, and whole-cell voltage-clamp techniques. Results: After long-term epileptic seizures, both sodium channel voltage-dependence of activation and steady-state inactivation shifted to more hyperpolarizing potentials, which resulted in the enlarged window current; the membrane density of sodium current decreased and the time constant of recovery from inactivation increased. CBZ displayed unchanged efficacy on sodium channels, with a similar binding rate to them, except that at higher concentrations, the voltage shift of inactivation was reduced. For the short-term kainate model rats, no differences were detected between the control and epilepsy groups. Conclusion: These results indicate that the properties of sodium channels in acutely dissociated hippocampal neurons could be changed following long-term epilepsy, but the alternation might not be enough to induce the channel resistance to CBZ.

  10. Excitability constraints on voltage-gated sodium channels.

    Directory of Open Access Journals (Sweden)

    Elaine Angelino

    2007-09-01

    Full Text Available We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.

  11. Sodium Channel (Dys)Function and Cardiac Arrhythmias

    NARCIS (Netherlands)

    C.A. Remme; C.R. Bezzina

    2010-01-01

    P>Cardiac voltage-gated sodium channels are transmembrane proteins located in the cell membrane of cardiomyocytes. Influx of sodium ions through these ion channels is responsible for the initial fast upstroke of the cardiac action potential. This inward sodium current thus triggers the initiation an

  12. Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels

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    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available Voltage-gated sodium channels (Nav are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the voltage-gated sodium channels. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

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

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    Radovanovic Aleksandar

    2008-12-01

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

  14. Pyrethroids differentially alter voltage-gated sodium channels from the honeybee central olfactory neurons.

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    Aklesso Kadala

    Full Text Available The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central 'antennal lobe neurons' (ALNs in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1 an acceleration of cumulative inactivation, and (2 a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and

  15. Elevated neuronal excitability due to modulation of the voltage-gated sodium channel Nav1.6 by Aβ1-42

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    Xi eWang

    2016-03-01

    Full Text Available Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer’s disease (AD. However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1-42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1-42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1-42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1-42. These results may illuminate the mechanism of aberrant neuronal networks in AD.

  16. Epithelial sodium channel modulates platelet collagen activation.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Benítez-Cardoza, Claudia; Ortega, Arturo

    2014-03-01

    Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation.

  17. Differential effects of TipE and a TipE-homologous protein on modulation of gating properties of sodium channels from Drosophila melanogaster.

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    Lingxin Wang

    Full Text Available β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4 in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNav9-1, DmNav22, and DmNav26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNav26. Further analysis of the gating kinetics of DmNav9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.

  18. PROPERTIES OF VOLTAGE-GATED SODIUM CHANNELS IN DEVELOPING AUDITORY NEURONS OF THE MOUSE IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective. To investigate the properties of voltage-gated sodium (Na+) channels in developing auditoryneurons during early postnatal stages in the mammalian central nervous system.Methods. Using the whole-cell voltage-clamp technique, we have studied changes in the electrophysi-ological properties of Na+ channels in the principal neurons of the medial nucleus of the trapezoid body (MNTB).Results. We found that MNTB neurons already express functional Na+ channels at postnatal day 1 (P1),and that channel density begins to increase at P5 when the neurons receive synaptic innervation andreach its maximum (~3 fold) at P11 when functional hearing onsets. These changes were paralleled byan age-dependent acceleration in both inactivation and recovery from inactivation. In contrast, there wasvery little alteration in the voltage-dependence of inactivation.Conclusion. These profound changes in the properties of voltage-gated Na+ channels may increase theexcitability of MNTB neurons and enhance their phase-locking fidelity and capacity during high-frequencysynaptic transmission.

  19. Voltage-gated sodium channels: mutations, channelopathies and targets.

    Science.gov (United States)

    Andavan, G S B; Lemmens-Gruber, R

    2011-01-01

    Voltage-gated sodium channels produce fast depolarization, which is responsible for the rising phase of the action potential in neurons, muscles and heart. These channels are very large membrane proteins and are encoded by ten genes in mammals. Sodium channels are a crucial component of excitable tissues; hence, they are a target for various neurotoxins that are produced by plants and animals for defence and protection, such as tetrodotoxin, scorpion toxins and batrachotoxin. Several mutations in various sodium channel subtypes cause multiple inherited diseases known as channelopathies. When these mutated sodium channel subtypes are expressed in various tissues, channelopathies in brain, skeletal muscle and cardiac muscle develop as well as neuropathic pain. In this review, we discuss aspects of voltage-gated sodium channel genes with an emphasis on cardiac muscle sodium channels. In addition, we report novel mutations that underlie a spectrum of diseases, such as Brugada, long QT syndrome and inherited conduction disorders. Furthermore, this review explains commonalities and differences among the channel subtypes, the channelopathies caused by the sodium channel gene mutation and the specificity of toxins and blockers of the channel subtypes.

  20. Neutralization of Gating Charges in Domain II of the Sodium Channel α Subunit Enhances Voltage-Sensor Trapping by a β-Scorpion Toxin

    Science.gov (United States)

    Cestèle, Sandrine; Scheuer, Todd; Mantegazza, Massimo; Rochat, Hervé; Catterall, William A.

    2001-01-01

    β-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently

  1. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Kozuka, M.; Ito, T.; Hirose, S.; Takahashi, K.; Hagiwara, H.

    1989-02-28

    Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using /sup 125/I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

  2. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

    Science.gov (United States)

    Bende, Niraj S; Kang, Eunji; Herzig, Volker; Bosmans, Frank; Nicholson, Graham M; Mobli, Mehdi; King, Glenn F

    2013-05-15

    One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Yuzhe Du

    2016-10-01

    Full Text Available Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr, in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel.

  4. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Zhorov, Boris S.; Dong, Ke

    2016-01-01

    Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr), in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel. PMID:27809228

  5. Transient Receptor Potential Vanilloid 4-Induced Modulation of Voltage-Gated Sodium Channels in Hippocampal Neurons.

    Science.gov (United States)

    Hong, Zhiwen; Jie, Pinghui; Tian, Yujing; Chen, Tingting; Chen, Lei; Chen, Ling

    2016-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is reported to control the resting membrane potential and increase excitability in many types of cells. Voltage-gated sodium channels (VGSCs) play an important role in initiating action potentials in neurons. However, whether VGSCs can be modulated by the activation of TRPV4 in hippocampal pyramidal neurons remains unknown. In this study, we tested the effect of TRPV4 agonists (GSK1016790A and 4α-PDD) on voltage-gated sodium current (I Na) in hippocampal CA1 pyramidal neurons and the protein levels of α/β-subunit of VGSCs in the hippocampus of mice subjected to intracerebroventricular (icv.) injection of GSK1016790A (GSK-injected mice). Herein, we report that I Na was inhibited by acute application of GSK1016790A or 4α-PDD. In the presence of TRPV4 agonists, the voltage-dependent inactivation curve shifted to the hyperpolarization, whereas the voltage-dependent activation curve remained unchanged. The TRPV4 agonist-induced inhibition of I Na was blocked by the TRPV4 antagonist or tetrodotoxin. Moreover, blocking protein kinase A (PKA) markedly attenuated the GSK1016790A-induced inhibition of I Na, whereas antagonism of protein kinase C or p38 mitogen-activated protein kinase did not change GSK1016790A action. Finally, the protein levels of Nav1.1, Nav1.2, and Nav1.6 in the hippocampus increased in GSK-injected mice, whereas those of Nav1.3 and Navβ1 remained nearly unchanged. We conclude that I Na is inhibited by the acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons, but protein expression of α-subunit of VGSCs is increased by sustained TRPV4 activation, which may compensate for the acute inhibition of I Na and provide a possibility for hyper-excitability upon sustained TRPV4 activation.

  6. Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

    Science.gov (United States)

    Jo, Sooyeon; Bean, Bruce P

    2017-04-01

    Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding.

  7. NALCN ion channels have alternative selectivity filters resembling calcium channels or sodium channels.

    Directory of Open Access Journals (Sweden)

    Adriano Senatore

    Full Text Available NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE or sodium channels (EKEE or EEKE. NALCN channels with alternative calcium, (EEEE and sodium, (EKEE or EEKE -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG(+ impermeant and blockable with 10 µM Gd(3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2:371-83.

  8. S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

    Science.gov (United States)

    Groome, James R; Winston, Vern

    2013-05-01

    The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

  9. Pertussis toxin modulation of sodium channels in the central neurons of cyhalothrin-resistant and cyhalothrin-susceptible cotton bollworm, Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    QIANG ZHAO; DE-LING KONG; BING-JUN HE; YAN-QIANG LIU; XIAN-LIN FAN; AN-XI LIU

    2007-01-01

    Pertussis toxin (PTX) inhibits the activation of the α-subunit of the inhibitory heterotrimeric G-proteins (Gαi/o) and modulates voltage-gated sodium channels, which may be one of the primary targets of pyrethroids. To investigate the potential mechanisms of agricultural pests resistance to pyrethroid insecticides, we examined the modulations by PTX on sodium channels in the central neurons of the 3rd-4th instar larvae of cyhalothrin-resistant (Cy-R) and cyhalothrin-susceptible (Cy-S) Helicoverpa armigera by the whole-cell patch-clamp technique.The isolated neurons were cultured for 12-16 h in an improved L15 insect culture medium with or without PTX (400 ng/mL). The results showed that both the Cy-R and Cy-S sodium channels exhibited fast kinetics and tetrodotoxin (TTX) sensitivity. The Cy-R sodium channels exhibited not only altered gating properties, including a 8.88-mV right shift in voltage-dependent activation (V0.5act) and a 6.54-mV right shift in voltage-dependent inactivation (V0.5inact), but also a reduced peak in sodium channel density (Idensity) (55.2% of that in Cy-S neurons). Cy-R sodium channels also showed low excitability, as evidenced by right shift of activation potential (Vacti) by 5-10 mV and peak potential (Vpeak) by 20 mV. PTX exerted significant effects on Cy-S sodium channels,reducing sodium channel density by 70.04%, right shifting V0.5act by 14.41 mV and V0.5inact by 9.38 mV. It did not cause any significant changes of the parameters mentioned above in the Cy-R sodium channels. The activation time (Tpeak) from latency to peak at peak voltage and the fast inactivation time constant (τinact) in both Cy-S and Cy-R neurons were not affected. The results suggest that cotton bollworm resistant to pyrethroid insecticides involves not only mutations and allosteric alterations of voltage-gated sodium channels, but also might implicate perturbation of PTX-sensitive Gαi/o-coupled signaling transduction pathways.

  10. Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones.

    Science.gov (United States)

    Power, Kevin E; Carlin, Kevin P; Fedirchuk, Brent

    2012-03-01

    Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of

  11. Simultaneous modifications of sodium channel gating by two scorpion toxins.

    OpenAIRE

    1982-01-01

    The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan ...

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

    Science.gov (United States)

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

    2010-06-01

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

  13. Native pyroglutamation of huwentoxin-IV: a post-translational modification that increases the trapping ability to the sodium channel.

    Directory of Open Access Journals (Sweden)

    Mingqiang Rong

    Full Text Available Huwentoxin-IV (HWTX-IV, a tetrodotoxin-sensitive (TTX-s sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV, having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms, mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms. Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.

  14. Ion selectivity strategies of sodium channel selectivity filters.

    Science.gov (United States)

    Dudev, Todor; Lim, Carmay

    2014-12-16

    CONSPECTUS: Sodium ion channels selectively transport Na(+) cations across the cell membrane. These integral parts of the cell machinery are implicated in regulating the cardiac, skeletal and smooth muscle contraction, nerve impulses, salt and water homeostasis, as well as pain and taste perception. Their malfunction often results in various channelopathies of the heart, brain, skeletal muscles, and lung; thus, sodium channels are key drug targets for various disorders including cardiac arrhythmias, heart attack, stroke, migraine, epilepsy, pain, cancer, and autoimmune disorders. The ability of sodium channels to discriminate the native Na(+) among other competing ions in the surrounding fluids is crucial for proper cellular functions. The selectivity filter (SF), the narrowest part of the channel's open pore, lined with amino acid residues that specifically interact with the permeating ion, plays a major role in determining Na(+) selectivity. Different sodium channels have different SFs, which vary in the symmetry, number, charge, arrangement, and chemical type of the metal-ligating groups and pore size: epithelial/degenerin/acid-sensing ion channels have generally trimeric SFs lined with three conserved neutral serines and/or backbone carbonyls; eukaryotic sodium channels have EKEE, EEKE, DKEA, and DEKA SFs with an invariant positively charged lysine from the second or third domain; and bacterial voltage-gated sodium (Nav) channels exhibit symmetrical EEEE SFs, reminiscent of eukaryotic voltage-gated calcium channels. How do these different sodium channel SFs achieve high selectivity for Na(+) over its key rivals, K(+) and Ca(2+)? What factors govern the metal competition in these SFs and which of these factors are exploited to achieve Na(+) selectivity in the different sodium channel SFs? The free energies for replacing K(+) or Ca(2+) bound inside different model SFs with Na(+), evaluated by a combination of density functional theory and continuum dielectric

  15. Pharmacological kinetics of BmK AS, a sodium channel site 4-specific modulator on Nav1.3

    Institute of Scientific and Technical Information of China (English)

    Zhi-Rui Liu; Jie Tao; Bang-Qian Dong; Gang Ding; Zhi-Jun Cheng; Hui-Qiong He; Yong-Hua Ji

    2012-01-01

    [Objective] In this study,the pharmacological kinetics of Buthus martensi Karsch (BmK) AS,a specific modulator of voltage-gated sodium channel site 4,was investigated on Nav1.3 expressed in Xenopus oocytes.[Methods] Twoelectrode voltage clamp was used to record the whole-cell sodium current.[Results] The peak currents of Nav1.3 were depressed by BmK AS over a wide range of concentrations (10,100,and 500 nmol/L).Most remarkably,BmK AS at 100 nmol/L hyperpolarized the voltage-dependence and increased the voltage-sensitivity of steady-state activation/inactivation.In addition,BmK AS was capable of hyperpolarizing not only the fast inactivation but also the slow inactivation,with a greater preference for the latter.Moreover,BmK AS accelerated the time constant and increased the ratio of recovery in Nav1.3 at all concentrations.[Conclusion]This study provides direct evidence that BmK AS facilitates steady-state activation and inhibits slow inactivation by stabilizing both the closed and open states of the Nav 1.3 channel,which might Result from an integrative binding to two receptor sites on the voltage-gated sodium channels.These [Results]may shed light on therapeutics against Nav1.3-targeted pathology.

  16. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding

    Science.gov (United States)

    1987-01-01

    sodium inhibitory effect of ethanol on channel - mediated sodium influx channels ...Exprnmantal Trherpeutics Ped in I.SA. Actions of Ethanol on Voltage-Sensitive Sodium Channels : Effects on Neurotoxin Binding1 MICHAEL J. MULLIN 2 and... sodium channels . This indirect allosteric mechanism for inhibition of [H]BTX-B binding. effect orethanol was concentration-dependent and was

  17. The sodium channel as a target for local anesthetic drugs

    Directory of Open Access Journals (Sweden)

    Harry A Fozzard

    2011-11-01

    Full Text Available Na channels are the source of excitatory currents for the nervous system and muscle. They are the target for a class of drugs called local anesthetics (LA, which have been used for local and regional anesthesia and for excitatory dysfunction problems such as epilepsy and cardiac arrhythmia. LA drugs are prototypes for new analgesic drugs. The LA drug binding site has been localized to the inner pore of the channel, where drugs interact mainly with a phenylalanine in domain IV S6. Drug affinity is both voltage- and use-dependent. Voltage-dependency is the result of changes in the conformation of the inner pore during channel activation and opening, allowing high energy interaction of drugs with the phenylalanine. LA drugs also reduce the gating current of Na channels, which represents the movement of charged residues in the voltage sensors. Specifically, drug binding to phenylalanine locks the domain III S4 in its outward (activated position, and slows recovery of the domain IV S4. Although strongly affecting gating, LA drugs almost certainly also block by steric occlusion of the pore. Molecular definition of the binding and blocking interactions may help in new drug development.

  18. Sodium metabisulfite modulation of potassium channels in pain-sensing dorsal root ganglion neurons.

    Science.gov (United States)

    Nie, Aifang; Wei, Cailing; Meng, Ziqiang

    2009-12-01

    The effects of sodium metabisulfite (SMB), a general food preservative, on potassium currents in rat dorsal root ganglion (DRG) neurons were investigated using the whole-cell patch-clamp technique. SMB increased the amplitudes of both transient outward potassium currents and delayed rectifier potassium current in concentration- and voltage-dependent manner. The transient outward potassium currents (TOCs) include a fast inactivating (A-current or IA) current and a slow inactivating (D-current or ID) current. SMB majorly increased IA, and ID was little affected. SMB did not affect the activation process of transient outward currents (TOCs), but the inactivation curve of TOCs was shifted to more positive potentials. The inactivation time constants of TOCs were also increased by SMB. For delayed rectifier potassium current (IK), SMB shifted the activation curve to hyperpolarizing direction. SMB differently affected TOCs and IK, its effects major on A-type K+ channels, which play a role in adjusting pain sensitivity in response to peripheral redox conditions. SMB did not increase TOCs and IK when adding DTT in pipette solution. These results suggested that SMB might oxidize potassium channels, which relate to adjusting pain sensitivity in pain-sensing DRG neurons.

  19. The Epithelial Sodium Channel and the Processes of Wound Healing

    Directory of Open Access Journals (Sweden)

    Silvia Chifflet

    2016-01-01

    Full Text Available The epithelial sodium channel (ENaC mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.

  20. Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation.

    Science.gov (United States)

    Nicholson, G M; Walsh, R; Little, M J; Tyler, M I

    1998-06-01

    The present study investigates the actions of robustoxin (atracotoxin-Ar1) purified from the venom of the male Sydney funnel-web spider Atrax robustus on sodium channel gating. Using whole-cell patch-clamp techniques the study assessed the actions of robustoxin on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in rat dorsal root ganglion cells. Similar to the closely related funnel-web spider toxin versutoxin (delta-atracotoxin-Hv1) from Hadronyche versuta, robustoxin had no effect on TTX-R sodium currents but exerted potent effects on TTX-S sodium currents. The main action of robustoxin was a concentration-dependent slowing or removal of TTX-S sodium current inactivation. This steady-state current was maintained during long-lasting depolarisations at all test potentials. Robustoxin (30 nM) also caused a 13-mV hyperpolarising shift in the voltage midpoint of steady-state sodium channel inactivation (h infinity) leading to a reduced peak current at a holding potential of -80 mV. Moreover there was a steady-state or non-inactivating component present (18% of maximal sodium current) at prepulse potentials that normally inactivate all TTX-S sodium channels (more depolarised than -40 mV). In addition robustoxin produced a significant increase in the repriming kinetics of the sodium channel when channels returned to the resting state following activation. This increase in the rate of recovery of sodium current appears to explain the use-dependent effects on peak sodium current amplitude at high stimulation frequencies. Finally 30 nM robustoxin caused an 11-mV hyperpolarising shift in the voltage dependence of the channel but did not markedly modify tail current kinetics. These actions suggest that robustoxin inhibits conversion of the open state to the inactivated state of TTX-S sodium channels, thus allowing a fraction of the sodium current to remain at membrane potentials at which inactivation is normally complete. Given the recent

  1. Sodium Channels, Mitochondria, and Axonal Degeneration in Peripheral Neuropathy.

    Science.gov (United States)

    Persson, Anna-Karin; Hoeijmakers, Janneke G J; Estacion, Mark; Black, Joel A; Waxman, Stephen G

    2016-05-01

    Peripheral neuropathy results from damage to peripheral nerves and is often accompanied by pain in affected limbs. Treatment represents an unmet medical need and a thorough understanding of the mechanisms underlying axonal injury is needed. Longer nerve fibers tend to degenerate first (length-dependence), and patients carrying pathogenic mutations throughout life usually become symptomatic in mid- or late-life (time-dependence). The activity of voltage-gated sodium channels can contribute to axonal injury and sodium channel gain-of-function mutations have been linked to peripheral neuropathy. Recent studies have implicated sodium channel activity, mitochondrial compromise, and reverse-mode Na(+)/Ca(2+) exchange in time- and length-dependent axonal injury. Elucidation of molecular mechanisms underlying axonal injury in peripheral neuropathy may provide new therapeutic strategies for this painful and debilitating condition.

  2. Physiological regulation of epithelial sodium channel by proteolysis

    DEFF Research Database (Denmark)

    Svenningsen, Per; Friis, Ulla G; Bistrup, Claus

    2011-01-01

    PURPOSE OF REVIEW: Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric...... disease. RECENT FINDINGS: A variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved α and γ-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1...... opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets....

  3. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    Science.gov (United States)

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Actions of Ethanol on Voltage-Sensitive Sodium Channels. Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    Science.gov (United States)

    1985-01-01

    concentration in the nonaqueuus (membrane) phase (Lyon et aL, 1981). Concentration- effect summarized in table 1 . When sodium channels were activated curves were...Voltage-Sensitive Sodium Channels : Effects on Neurotoxin-Stimulated Sodium Uptake in DT (7 Synaptosomes E L C MICHAEL J. MULLIN’ and WALTER A. HUNT...1984). At the present time, the 8 1 structural and functional properties of the voltage-sensitive sodium channels are understood most completely

  5. Channelopathies from Mutations in the Cardiac Sodium Channel Protein Complex

    Science.gov (United States)

    Adsit, Graham S.; Vaidyanathan, Ravi; Galler, Carla M.; Kyle, John W.; Makielski, Jonathan C.

    2013-01-01

    The cardiac sodium current underlies excitability in heart, and inherited abnormalities of the proteins regulating and conducting this current cause inherited arrhythmia syndromes. This review focuses on inherited mutations in non-pore forming proteins of sodium channel complexes that cause cardiac arrhythmia, and the deduced mechanisms by which they affect function and dysfunction of the cardiac sodium current. Defining the structure and function of these complexes and how they are regulated will contribute to understanding the possible roles for this complex in normal and abnormal physiology and homeostasis. PMID:23557754

  6. Channelopathies from mutations in the cardiac sodium channel protein complex.

    Science.gov (United States)

    Adsit, Graham S; Vaidyanathan, Ravi; Galler, Carla M; Kyle, John W; Makielski, Jonathan C

    2013-08-01

    The cardiac sodium current underlies excitability in heart, and inherited abnormalities of the proteins regulating and conducting this current cause inherited arrhythmia syndromes. This review focuses on inherited mutations in non-pore forming proteins of sodium channel complexes that cause cardiac arrhythmia, and the deduced mechanisms by which they affect function and dysfunction of the cardiac sodium current. Defining the structure and function of these complexes and how they are regulated will contribute to understanding the possible roles for this complex in normal and abnormal physiology and homeostasis. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. [Progress in sodium channelopathies and biological functions of voltage-gated sodium channel blockers].

    Science.gov (United States)

    Wang, Hongyan; Gou, Meng; Xiao, Rong; Li, Qingwei

    2014-06-01

    Voltage-gated sodium channels (VGSCs), which are widely distributed in the excitable cells, are the primary mediators of electrical signal amplification and propagation. They play important roles in the excitative conduction of the neurons and cardiac muscle cells. The abnormalities of the structures and functions of VGSCs can change the excitability of the cells, resulting in a variety of diseases such as neuropathic pain, epilepsy and arrhythmia. At present, some voltage-gated sodium channel blockers are used for treating those diseases. In the recent years, several neurotoxins have been purified from the venom of the animals, which could inhibit the current of the voltage-gated sodium channels. Usually, these neurotoxins are compounds or small peptides that have been further designed and modified for targeted drugs of sodium channelopathies in the clinical treatment. In addition, a novel cysteine-rich secretory protein (CRBGP) has been isolated and purified from the buccal gland of the lampreys (Lampetra japonica), and it could inhibit the Na+ current of the hippocampus and dorsal root neurons for the first time. In the present study, the progress of the sodium channelopathies and the biological functions of voltage-gated sodium channel blockers are analyzed and summarized.

  8. Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1.

    Science.gov (United States)

    Inserra, Marco C; Israel, Mathilde R; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R; Harrington, Andrea M; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y; Zimmermann, Katharina; Lewis, Richard J; Brierley, Stuart M; Vetter, Irina

    2017-02-22

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

  9. Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

    Science.gov (United States)

    Inserra, Marco C.; Israel, Mathilde R.; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R.; Harrington, Andrea M.; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y.; Zimmermann, Katharina; Lewis, Richard J.; Brierley, Stuart M.; Vetter, Irina

    2017-01-01

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation. PMID:28225079

  10. Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals

    Directory of Open Access Journals (Sweden)

    Gasser Andreas

    2010-11-01

    Full Text Available Abstract Background Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals. Results Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro. Conclusions NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

  11. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.

    Science.gov (United States)

    Wu, Min-Tzu; Huang, Po-Yuan; Yen, Chen-Tung; Chen, Chih-Cheng; Lee, Ming-Jen

    2013-01-01

    Primary erythromelalgia (PE) is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or exercise. Mutations in human SCN9A gene, encoding the α-subunit of the voltage-gated sodium channel, Na(v)1.7, were found to be responsible for PE. Three missense mutations of SCN9A gene have recently been identified in Taiwanese patients including a familial (I136V) and two sporadic mutations (I848T, V1316A). V1316A is a novel mutation and has not been characterized yet. Topologically, I136V is located in DI/S1 segment and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the elelctrophysiological manifestations, the channel conductance with whole-cell patch clamp was recorded on the over-expressed Chinese hamster overy cells. As compared with wild type, the mutant channels showed a significant hyperpolarizing shift in voltage dependent activation and a depolarizing shift in steady-state fast inactivation. The recovery time from channel inactivation is faster in the mutant than in the wild type channels. Since warmth can trigger and exacerbate symptoms, we then examine the influence of tempearture on the sodium channel conduction. At 35°C, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift at activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. High temperature caused a significant depolarizing shift in steady-state fast inactivation in all three mutant channels. These findings may confer to the hyperexcitability of sensory neurons, especially at high temperature. In order to identifying an effective treatment, we tested the IC₅₀ values of selective sodium channel blockers, lidocaine and mexiletine. The IC₅₀ for mexiletine is lower for I848T mutant channel as compared to that of the wild type and other two

  12. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.

    Directory of Open Access Journals (Sweden)

    Min-Tzu Wu

    Full Text Available Primary erythromelalgia (PE is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or exercise. Mutations in human SCN9A gene, encoding the α-subunit of the voltage-gated sodium channel, Na(v1.7, were found to be responsible for PE. Three missense mutations of SCN9A gene have recently been identified in Taiwanese patients including a familial (I136V and two sporadic mutations (I848T, V1316A. V1316A is a novel mutation and has not been characterized yet. Topologically, I136V is located in DI/S1 segment and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the elelctrophysiological manifestations, the channel conductance with whole-cell patch clamp was recorded on the over-expressed Chinese hamster overy cells. As compared with wild type, the mutant channels showed a significant hyperpolarizing shift in voltage dependent activation and a depolarizing shift in steady-state fast inactivation. The recovery time from channel inactivation is faster in the mutant than in the wild type channels. Since warmth can trigger and exacerbate symptoms, we then examine the influence of tempearture on the sodium channel conduction. At 35°C, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift at activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. High temperature caused a significant depolarizing shift in steady-state fast inactivation in all three mutant channels. These findings may confer to the hyperexcitability of sensory neurons, especially at high temperature. In order to identifying an effective treatment, we tested the IC₅₀ values of selective sodium channel blockers, lidocaine and mexiletine. The IC₅₀ for mexiletine is lower for I848T mutant channel as compared to that of the wild type

  13. A sodium-channel mutation causes isolated cardiac conduction disease

    NARCIS (Netherlands)

    Tan, HL; Bink-Boelkens, MTE; Bezzina, CR; Viswanathan, PC; Beaufort-Krol, GCM; van Tintelen, PJ; van den Berg, MP; Wilde, AAM; Balser, [No Value

    2001-01-01

    Cardiac conduction disorders slow the heart rhythm and cause disability in millions of people worldwide. Inherited mutations in SCN5A, the gene encoding the human cardiac sodium (Na+) channel, have been associated with rapid heart rhythms that occur suddenly and are life-threatening(1-3); however, a

  14. Development of a voltage-dependent current noise algorithm for conductance-based stochastic modelling of auditory nerve fibres.

    Science.gov (United States)

    Badenhorst, Werner; Hanekom, Tania; Hanekom, Johan J

    2016-12-01

    This study presents the development of an alternative noise current term and novel voltage-dependent current noise algorithm for conductance-based stochastic auditory nerve fibre (ANF) models. ANFs are known to have significant variance in threshold stimulus which affects temporal characteristics such as latency. This variance is primarily caused by the stochastic behaviour or microscopic fluctuations of the node of Ranvier's voltage-dependent sodium channels of which the intensity is a function of membrane voltage. Though easy to implement and low in computational cost, existing current noise models have two deficiencies: it is independent of membrane voltage, and it is unable to inherently determine the noise intensity required to produce in vivo measured discharge probability functions. The proposed algorithm overcomes these deficiencies while maintaining its low computational cost and ease of implementation compared to other conductance and Markovian-based stochastic models. The algorithm is applied to a Hodgkin-Huxley-based compartmental cat ANF model and validated via comparison of the threshold probability and latency distributions to measured cat ANF data. Simulation results show the algorithm's adherence to in vivo stochastic fibre characteristics such as an exponential relationship between the membrane noise and transmembrane voltage, a negative linear relationship between the log of the relative spread of the discharge probability and the log of the fibre diameter and a decrease in latency with an increase in stimulus intensity.

  15. Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

    Energy Technology Data Exchange (ETDEWEB)

    Goetz, R.; Dover, K; Laezza, F; Shtraizent, N; Huang, X; Tchetchik, D; Eliseenkova, A; Goldfarb, M; Mohammadi, M; et. al.

    2009-01-01

    Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.

  16. Regulation of Lung Epithelial Sodium Channels by Cytokines and Chemokines

    Directory of Open Access Journals (Sweden)

    Brandi M. Wynne

    2017-07-01

    Full Text Available Acute lung injury leading to acute respiratory distress (ARDS is a global health concern. ARDS patients have significant pulmonary inflammation leading to flooding of the pulmonary alveoli. This prevents normal gas exchange with consequent hypoxemia and causes mortality. A thin fluid layer in the alveoli is normal. The maintenance of this thin layer results from fluid movement out of the pulmonary capillaries into the alveolar interstitium driven by vascular hydrostatic pressure and then through alveolar tight junctions. This is then balanced by fluid reabsorption from the alveolar space mediated by transepithelial salt and water transport through alveolar cells. Reabsorption is a two-step process: first, sodium enters via sodium-permeable channels in the apical membranes of alveolar type 1 and 2 cells followed by active extrusion of sodium into the interstitium by the basolateral Na+, K+-ATPase. Anions follow the cationic charge gradient and water follows the salt-induced osmotic gradient. The proximate cause of alveolar flooding is the result of a failure to reabsorb sufficient salt and water or a failure of the tight junctions to prevent excessive movement of fluid from the interstitium to alveolar lumen. Cytokine- and chemokine-induced inflammation can have a particularly profound effect on lung sodium transport since they can alter both ion channel and barrier function. Cytokines and chemokines affect alveolar amiloride-sensitive epithelial sodium channels (ENaCs, which play a crucial role in sodium transport and fluid reabsorption in the lung. This review discusses the regulation of ENaC via local and systemic cytokines during inflammatory disease and the effect on lung fluid balance.

  17. Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels.

    Science.gov (United States)

    Sula, Altin; Wallace, B A

    2017-06-05

    Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation. © 2017 Sula and Wallace.

  18. 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.

  19. Hypotonicity activates a voltage-dependent membrane conductance in N2a neuroblastoma cells.

    Science.gov (United States)

    Taruno, Akiyuki; Marunaka, Yoshinori

    2017-03-04

    To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (Id) is insensitive to NPPB, a nonspecific blocker of Cl(-) channels, and intracellular Mg(2+), which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na(+) with NMDG(+) reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells.

  20. Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies

    Directory of Open Access Journals (Sweden)

    Eleonora eSavio Galimberti

    2012-07-01

    Full Text Available Voltage-gated sodium channels (VGSC are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa and can also function as cell-adhesion molecules (CAMs. In-vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named channel partners or channel interacting proteins (ChiPs like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin, and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics. Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium channelopathies. This review will outline the structure, function and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field

  1. Voltage-Gated Sodium Channels: Biophysics, Pharmacology, and Related Channelopathies

    Science.gov (United States)

    Savio-Galimberti, Eleonora; Gollob, Michael H.; Darbar, Dawood

    2012-01-01

    Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named “channel partners” or “channel interacting proteins” (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium “channelopathies”.  This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field. PMID:22798951

  2. Structure of membrane-active toxin from crab spider Heriaeus melloteei suggests parallel evolution of sodium channel gating modifiers in Araneomorphae and Mygalomorphae.

    Science.gov (United States)

    Berkut, Antonina A; Peigneur, Steve; Myshkin, Mikhail Yu; Paramonov, Alexander S; Lyukmanova, Ekaterina N; Arseniev, Alexander S; Grishin, Eugene V; Tytgat, Jan; Shenkarev, Zakhar O; Vassilevski, Alexander A

    2015-01-01

    We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

  3. Mapping of scorpion toxin receptor sites at voltage-gated sodium channels.

    Science.gov (United States)

    Gurevitz, Michael

    2012-09-15

    Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain

  4. A structural and dynamic molecular model for the sodium channel of Electrophorus electricus.

    Science.gov (United States)

    Kosower, E M

    1985-03-25

    Chemical logic and single group rotation (SGR) theory are applied to the primary structure determined by Noda et al. [(1984) Nature 312, 121-127] to construct a molecular model of the sodium channel of Electrophorus electricus. Both structural and dynamic aspects of the channel are accounted for, including gating current, sensitivity to changes in membrane potential, channel opening, a binding site for sodium, selectivity for sodium over potassium, capacity for rapid sodium flow, sensitivity to batrachotoxin (or other toxins) and inactivation.

  5. Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels.

    Science.gov (United States)

    Irie, Katsumasa; Kitagawa, Kazuya; Nagura, Hitoshi; Imai, Tomoya; Shimomura, Takushi; Fujiyoshi, Yoshinori

    2010-02-05

    Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

  6. Glutamine substitution at alanine1649 in the S4-S5 cytoplasmic loop of domain 4 removes the voltage sensitivity of fast inactivation in the human heart sodium channel.

    Science.gov (United States)

    Tang, L; Chehab, N; Wieland, S J; Kallen, R G

    1998-05-01

    Normal activation-inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4-S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89-104.). We now show that glutamine substitutions NH2-terminal to the methionines (L1646, L1647, F1648, A1649, L1650) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4-S5/D4 segment (L1654, P1655, A1656) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641-658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A1649 or Y1494Y1495 may disrupt a linkage between S4-S5/D4 and the interdomain 3-4 loop interfering with normal activation-inactivation coupling.

  7. Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1.

    Science.gov (United States)

    Briones, Rodolfo; Weichbrodt, Conrad; Paltrinieri, Licia; Mey, Ingo; Villinger, Saskia; Giller, Karin; Lange, Adam; Zweckstetter, Markus; Griesinger, Christian; Becker, Stefan; Steinem, Claudia; de Groot, Bert L

    2016-09-20

    The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.

  8. Molecular Mechanism that Leads to Development of Epilepsy by a Sodium Channel Mutation

    OpenAIRE

    Velazquez, Eric

    2014-01-01

    The number of mutations in the voltage-gated sodium channel Nav1.1, encoded by SCN1A, that have been associated with genetic epilepsy is extremely high compared to the other sodium channel genes. The crucial function of the voltage-gated sodium channels in the central nervous system (CNS) is to regulate the influx of sodium or sodium current into the neurons. Sodium currents are required for the generation and propagation of action potentials. Subsequently the action potentials cause the rele...

  9. The complete structure of an activated open sodium channel

    Science.gov (United States)

    Sula, Altin; Booker, Jennifer; Ng, Leo C. T.; Naylor, Claire E.; DeCaen, Paul G.; Wallace, B. A.

    2017-01-01

    Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4–S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate. PMID:28205548

  10. Adaptive evolution of the vertebrate skeletal muscle sodium channel

    Directory of Open Access Journals (Sweden)

    Jian Lu

    2011-01-01

    Full Text Available Tetrodotoxin (TTX is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na v. The skeletal muscle Na v (Na v1.4 channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na v1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na v1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na v1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.

  11. Marine Toxins That Target Voltage-gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Robert J. French

    2006-04-01

    Full Text Available Abstract: Eukaryotic, voltage-gated sodium (NaV channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different NaV receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt NaV channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10-70 amino acids. We review the variety of known NaV-targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.

  12. Voltage dependence of the Na-K pump.

    Science.gov (United States)

    De Weer, P; Gadsby, D C; Rakowski, R F

    1988-01-01

    Present evidence demonstrates that the Na-K pump rate is voltage dependent, whereas early work was largely inconclusive. The I-V relationship has a positive slope over a wide voltage range, and the existence of a negative slope region is now doubtful. Monotonic voltage dependence is consistent with the reaction cycle containing a single voltage-dependent step. Recent measurements suggest that this voltage-dependent step occurs during Na translocation and may be deocclusion of Na+. In addition, two results suggest that K translocation is voltage insensitive: (a) large positive potentials appear to have no influence on Rb-Rb exchange or associated conformational transitions; and (b) transient currents associated with Na translocation appear to involve movement of a single charge, which is sufficient for a 3Na-2K cycle. The simplest interpretation is that the pump's cation binding sites supply two negative charges. Pre-steady-state measurements demonstrate that Na translocation precedes the pump cycle's rate-limiting step, presumably K translocation. But, because K translocation seems voltage insensitive, the voltage dependence of the steady-state pump rate probably reflects that of the concentration of the intermediate entering this slow step. Further pump current and flux data (both transient and steady-state), carefully determined over a range of conditions, should increase our understanding of the voltage-dependent step(s) in the Na-K pump cycle.

  13. Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels.

    Science.gov (United States)

    Schiavon, Emanuele; Pedraza-Escalona, Martha; Gurrola, Georgina B; Olamendi-Portugal, Timoteo; Corzo, Gerardo; Wanke, Enzo; Possani, Lourival D

    2012-02-01

    The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na(v)1.1-Na(v)1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na(v)1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na(v)1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na+ -channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.

  14. Fibrinolytic Regulation of Pulmonary Epithelial Sodium Channels: a Critical Review

    OpenAIRE

    Ji, Hong-Long

    2015-01-01

    Luminal fluid homeostasis in the respiratory system is crucial to maintain the gas-\\ud blood exchange in normal lungs and mucociliary clearance in the airways. Epithelial\\ud sodium channels (ENaC) govern ~70% of alveolar fluid clearance. Four ENaC subunits\\ud have been cloned, namely, α, β, γ, and δ ENaC subunits in mammalian cells. This\\ud critical review focuses on the expression and function of ENaC in human and murine\\ud lungs, and the post-translational regulation by fibrinolysins. Nebul...

  15. Zn2+ regulates Kv2.1 voltage-dependent gating and localization following ischemia

    OpenAIRE

    Aras, Mandar A.; Saadi, Robert A.; Aizenman, Elias

    2009-01-01

    The delayed-rectifier K+ channel Kv2.1 exists in highly phosphorylated somatodendritic clusters. Ischemia induces rapid Kv2.1 dephosphorylation and a dispersal of these clusters, accompanied by a hyperpolarizing shift in their voltage-dependent activation kinetics. Transient modulation of Kv2.1 activity and localization following ischemia is dependent on a rise in intracellular Ca2+and the protein phosphatase calcineurin. Here, we show that neuronal free Zn2+also plays a critical role in the ...

  16. Sodium channel β subunits: emerging targets in channelopathies.

    Science.gov (United States)

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

  17. A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids.

    Science.gov (United States)

    Hu, Zhaonong; Du, Yuzhe; Nomura, Yoshiko; Dong, Ke

    2011-01-01

    Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the α-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.

  18. Epithelial Sodium and Acid-Sensing Ion Channels

    Science.gov (United States)

    Kellenberger, Stephan

    The epithelial Na+ channel (ENaC) and acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels that form their own subfamilies within the ENaC/degenerin ion channel family. ASICs are sensors of extracellular pH, and ENaC, whose main function is trans-epithelial Na+ transport, can sense extra- and intra-cellular Na+. In aldosterone-responsive epithelial cells of the kidney, ENaC plays a critical role in the control of sodium balance, blood volume and blood pressure. In airway epithelia, ENaC has a distinct role in controlling fluid reabsorption at the air-liquid interface, thereby determining the rate of mucociliary transport. In taste receptor cells of the tongue, ENaC is involved in salt taste sensation. ASICs have emerged as key sensors for extracellular protons in central and peripheral neurons. Although not all of their physiological and pathological functions are firmly established yet, there is good evidence for a role of ASICs in the brain in learning, expression of fear, and in neurodegeneration after ischaemic stroke. In sensory neurons, ASICs are involved in nociception and mechanosensation. ENaC and ASIC subunits share substantial sequence homology and the conservation of several functional domains. This chapter summarises our current understanding of the physiological functions and of the mechanisms of ion permeation, gating and regulation of ENaC and ASICs.

  19. Anticonvulsant and sodium channel blocking activity of higher doses of clenbuterol.

    Science.gov (United States)

    Fischer, W; Kittner, H; Regenthal, R; Malinowska, B; Schlicker, E

    2001-02-01

    of clenbuterol against generalized tonic-clonic and complex partial seizures seem to be related to the inhibition of voltage-dependent sodium channels and not to the modulation of beta-adrenoceptors.

  20. Controlling epithelial sodium channels with light using photoswitchable amilorides

    Science.gov (United States)

    Schönberger, Matthias; Althaus, Mike; Fronius, Martin; Clauss, Wolfgang; Trauner, Dirk

    2014-08-01

    Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane proteins, assembled from β, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx into non-epithelial cells. The functional role of δβγENaC in various organs, including the human brain, is still poorly understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical control of ENaC channels, in particular the δβγ isoform, by switching between blue and green light, or by turning on and off blue light. PA1 was used to modify functionally δβγENaC in amphibian and mammalian cells. We also show that PA1 can be used to differentiate between δβγENaC and αβγENaC in a model for the human lung epithelium.

  1. The enigmatic drug binding site for sodium channel inhibitors.

    Science.gov (United States)

    Mike, Arpad; Lukacs, Peter

    2010-11-01

    Local anesthetics have been in clinical use since 1884, and different aspects of the local anesthetic binding site have been studied in enormous detail. In spite of all these efforts, some of the most fundamental questions--such as which exact residues constitute the binding site, how many binding sites exist, do local anesthetics share their binding site(s) with other sodium channel inhibitors, and what are the mechanisms of inhibition--are still largely unanswered. We review accumulated data on the "local anesthetic receptor"and discuss controversial points, such as possible mechanisms of inhibition, the possibility of additional binding sites, the orientation of S6 helices, and the internal vs. external position of the anticonvulsant binding site. We describe the four following specific groups of functionally important residues: i) conserved asparagines six residues below the hinge residues; we propose that they are oriented toward the external surface of S6 helices, and have a critical role in the coupling of voltage sensors to gating, ii) residues lining the inner vestibule and constructing the "orthodox" binding site, iii) residues around the outer vestibule, which have been proposed to constitute an alternative external binding site, and iv) residues determining external access for quaternary amine inhibitors, such as QX314. We conclude that sodium channel inhibitors must be heterogenous in terms of binding sites and inhibition mechanisms, and propose that this heterogeneity should be taken into consideration during drug development.

  2. Rufinamide Improves Functional and Behavioral Deficits via Blockade of Tetrodotoxin-Resistant Sodium Channels in Diabetic Neuropathy.

    Science.gov (United States)

    Kharatmal, Shivsharan B; Singh, Jitendra N; Sharma, Shyam S

    2015-01-01

    Rufinamide is a structurally novel, antiepileptic drug approved for the treatment of Lennox-Gastaut syndrome. Its mechanism of action involves inhibition of voltage-gated Na+ channels (VGSCs) with possible membrane-stabilizing effects. VGSCs play a significant role in the pathogenesis of neuropathic pain. Therefore, we investigated the effects of rufinamide on tetrodotoxin-resistant sodium current (TTX-R I(Na)) in acutely dissociated rat dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats by using whole-cell voltage-clamp configuration. In addition, the functional and behavioural nociceptive parameters were evaluated to assess its potential in diabetic neuropathy. Diabetic rats demonstrated the mechanical allodynia and thermal hyperalgesia with reduced nerve perfusion and conduction velocity as compared to control. Rufinamide treatments (3 and 10 mg/kg) significantly improved these functional and nociceptive deficits. Diabetic rat DRG neurons exhibited increased TTX-R I(Na) density as compared to control. The voltage-dependent activation and steady-state inactivation curves for TTX-R I(Na) in DRG neurons from diabetic rats were shifted negatively as compared to control. Rufinamide treatments significantly blocked the TTX-R Na+ channel activity as evident from significant reduction in I(Na) density and hyperpolarizing shift in activation and inactivation curves as compared to diabetic control. This suggests that rufinamide acts on TTX-R Na+ channels, reduces channel activity and attenuates nerve functional and behavioral parameters in diabetic rats. Altogether, these results indicate therapeutic potential of rufinamide in the treatment of diabetic neuropathy.

  3. Non-specific activation of the epithelial sodium channel by the CFTR chloride channel

    OpenAIRE

    Nagel, Georg; Szellas, Tanjef; Riordan, John R.; Friedrich, Thomas; Hartung, Klaus

    2001-01-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of 22Na+ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride ...

  4. Voltage-dependent Calcium Channel Plays a Role in the Formation of Large-amplitude Miniature Excitatory Postsynaptic Current%电压依赖性钙通道参与大振幅微小兴奋性突触后电流形成的实验研究

    Institute of Scientific and Technical Information of China (English)

    黄福森; 杨小娟; 王儒蓉; 吴超然

    2012-01-01

    目的 观察电压依赖性钙通道是否作用于大鼠脊髓背角胶状质层(SG)神经元大振幅微小兴奋性突触后电流的形成.方法 选用成年雄性Sprague-Dawley (SD)大鼠,2%~3%异氟烷麻醉后,分离其腰骶部的脊髓,然后切片.采用全细胞电压钳技术,玻璃微电极的电阻为4~6 MΩ,钳制电压为-70 mV,记录胶状质层神经元微小兴奋性突触后电流( mEPSC)电流.将电流信号用Axopatch 200来放大并储存于电脑.对照组和用药结束后,持续采样mEPSC电流30 s.mEPSC电流的频率和振幅用Clampfit 8.1进行分析.结果 钳制电压为-70 mV时,所有SG神经元均有自发性的EPSC.辣椒素增加mEPSC发生的频率和波幅.钴离子抑制辣椒素诱导的大振幅mEPSC.钴离子抑制辣椒素诱导的mEPSC的平均振幅,而不抑制其发生频率.结论 电压依赖性钙离子通道参与了辣椒素引起的痛觉形成.%Objective To observe whether the voltage-dependent calcium channel contributes to the formation of capsaicin-induced miniature excitatory postsynaptic current (mEPSC) in rats. Methods Experiments were performed in adult male Sprague-Dawley rats. The lumbosacral portion of the spinal cord were separated after anesthesia by 2%-3% isoflurane, and the spinal cord slices were prepared. Whole-cell voltage-clamp technique was applied to substantia gelatinosa (SG) neurons with a glass patch-pipette having a resistance of 4-6 MΩ, holding potential -70 mV. Signals were amplified with an Axopatch 200 amplifier and then stored in a personal computer. The mEPSC in controls and immediately after the end of drug applications were sampled for 30 seconds and the frequency and amplitude were analyzed using Clampfit 8.1. Results All SG neurons in this database had spontaneous mEPSC with the holding potential of-70 mV. Capsaicin increased the frequency and mean amplitude of mEPSC. Cobalt inhibited the capsaicin-induced large-amplitude mEPSC, as well as the mean amplitude but

  5. Molecular model of the action potential sodium channel.

    Science.gov (United States)

    Guy, H R; Seetharamulu, P

    1986-01-01

    Secondary and tertiary structural models of sodium channel transmembrane segments were developed from its recently determined primary sequence in Electrophorus electricus. The model has four homologous domains, and each domain has eight homologous transmembrane segments, S1 through S8. Each domain contains three relatively apolar segments (S1, S2 and S3) and two very apolar segments (S5 and S8), all postulated to be transmembrane alpha-helices. S4 segments have positively charged residues, mainly arginines, at every third residue. The model channel lining is formed by four S4 transmembrane alpha-helices and four negatively charged S7 segments. S7 segments are postulated to be short, partially transmembrane amphipathic alpha-helices in three domains and a beta-strand in the last domain. S7 segments are preceded by short apolar segments (S6) postulated to be alpha-helices in three domains and a beta-strand in the last domain. Positively charged side chains of S4 form salt bridges with negatively charged side chains on S7 and near the ends of S1 and S3. Putative extracellular segments that contain 5 of the 10 potential N-glycosylation sites link S5 to S6. Channel activation may involve a 'helical screw' mechanism in which S4 helices rotate around their axes as they move toward the extracellular surface. Images PMID:2417247

  6. Antinociceptive Effects of AGAP, a Recombinant Neurotoxic Polypeptide: Possible Involvement of the Tetrodotoxin-Resistant Sodium Channels in Small Dorsal Root Ganglia Neurons

    Science.gov (United States)

    Li, Chun-Li; Liu, Xi-Fang; Li, Gui-Xia; Ban, Meng-qi; Chen, Jian-Zhao; Cui, Yong; Zhang, Jing-Hai; Wu, Chun-Fu

    2016-01-01

    Antitumor-analgesic peptide (AGAP) is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG) neurons. The results showed that AGAP (3–1000 nM) inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R) sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide. PMID:28066245

  7. Antinociceptive effects of AGAP, a recombinant neurotoxic polypeptide: Possible involvement of the tetrodotoxin-resistant sodium channels in small dorsal root ganglia neurons

    Directory of Open Access Journals (Sweden)

    Li Chunli

    2016-12-01

    Full Text Available Antitumor-analgesic peptide(AGAP)is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG neurons. The results showed that AGAP (3-1000 nM inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide.

  8. Differential state-dependent modification of rat Na{sub v}1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin

    Energy Technology Data Exchange (ETDEWEB)

    He, Bingjun [College of Life Sciences, Nankai University, Tianjin 300071 (China); Soderlund, David M., E-mail: dms6@cornell.edu [Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (United States)

    2011-12-15

    We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}1 and {beta}2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na{sub v}1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent 'late' currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were {approx} 25 mV for tefluthrin and {approx} 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of {approx} 5-10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na{sub v}1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by {approx} 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na{sub v}1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na{sub v}1.6 channels

  9. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

    Science.gov (United States)

    Bourdin, Céline M; Moignot, Bénédicte; Wang, Lingxin; Murillo, Laurence; Juchaux, Marjorie; Quinchard, Sophie; Lapied, Bruno; Guérineau, Nathalie C; Dong, Ke; Legros, Christian

    2013-01-01

    Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an

  10. Biophysical Adaptations of Prokaryotic Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Vien, T N; DeCaen, P G

    2016-01-01

    This chapter describes the adaptive features found in voltage-gated sodium channels (NaVs) of prokaryotes and eukaryotes. These two families are distinct, having diverged early in evolutionary history but maintain a surprising degree of convergence in function. While prokaryotic NaVs are required for growth and motility, eukaryotic NaVs selectively conduct fast electrical currents for short- and long-range signaling across cell membranes in mammalian organs. Current interest in prokaryotic NaVs is stoked by their resolved high-resolution structures and functional features which are reminiscent of eukaryotic NaVs. In this chapter, comparisons between eukaryotic and prokaryotic NaVs are made to highlight the shared and unique aspects of ion selectivity, voltage sensitivity, and pharmacology. Examples of prokaryotic and eukaryotic NaV convergent evolution will be discussed within the context of their structural features. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Plasmin in nephrotic urine activates the epithelial sodium channel

    DEFF Research Database (Denmark)

    Svenningsen, Per; Bistrup, Claus; Friis, Ulla G;

    2008-01-01

    Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus...... laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors...... of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen...

  12. The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate.

    Science.gov (United States)

    Mistry, Abinash C; Wynne, Brandi M; Yu, Ling; Tomilin, Viktor; Yue, Qiang; Zhou, Yiqun; Al-Khalili, Otor; Mallick, Rickta; Cai, Hui; Alli, Abdel A; Ko, Benjamin; Mattheyses, Alexa; Bao, Hui-Fang; Pochynyuk, Oleh; Theilig, Franziska; Eaton, Douglas C; Hoover, Robert S

    2016-10-01

    The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  13. Potassium current inhibition by nonselective cation channel-mediated sodium entry in rat pheochromocytoma (PC-12) cells.

    OpenAIRE

    Strübing, C; J Hescheler

    1996-01-01

    Under physiological conditions, nonselective cation (NSC) channels mediate the entry of cations into cells, the most important being Na+ and Ca2+. In contrast to the Ca(2+)-dependent signaling mechanisms, little is known about the consequences and the spatial distribution of intracellular [Na+] elevation. In this study we demonstrate that Na+ entry, during the opening of ATP-activated NSC channels, leads to an inhibition of voltage-dependent K+ currents (IK) in cromaffin-like undifferentiated...

  14. Blood pressure and amiloride-sensitive sodium channels in vascular and renal cells.

    Science.gov (United States)

    Warnock, David G; Kusche-Vihrog, Kristina; Tarjus, Antoine; Sheng, Shaohu; Oberleithner, Hans; Kleyman, Thomas R; Jaisser, Frederic

    2014-03-01

    Sodium transport in the distal nephron is mediated by epithelial sodium channel activity. Proteolytic processing of external domains and inhibition with increased sodium concentrations are important regulatory features of epithelial sodium channel complexes expressed in the distal nephron. By contrast, sodium channels expressed in the vascular system are activated by increased external sodium concentrations, which results in changes in the mechanical properties and function of endothelial cells. Mechanosensitivity and shear stress affect both epithelial and vascular sodium channel activity. Guyton's hypothesis stated that blood pressure control is critically dependent on vascular tone and fluid handling by the kidney. The synergistic effects, and complementary regulation, of the epithelial and vascular systems are consistent with the Guytonian model of volume and blood pressure regulation, and probably reflect sequential evolution of the two systems. The integration of vascular tone, renal perfusion and regulation of renal sodium reabsorption is the central underpinning of the Guytonian model. In this Review, we focus on the expression and regulation of sodium channels, and we outline the emerging evidence that describes the central role of amiloride-sensitive sodium channels in the efferent (vascular) and afferent (epithelial) arms of this homeostatic system.

  15. Molecular basis for class Ib anti-arrhythmic inhibition of cardiac sodium channels

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Galpin, Jason D; Frankel, Adam

    2011-01-01

    Cardiac sodium channels are established therapeutic targets for the management of inherited and acquired arrhythmias by class I anti-arrhythmic drugs (AADs). These drugs share a common target receptor bearing two highly conserved aromatic side chains, and are subdivided by the Vaughan...... the inhibition of cardiac sodium channels by clinically relevant drugs and provide information for the directed design of AADs....

  16. The sodium-leak channel, NALCN, in health and disease

    Directory of Open Access Journals (Sweden)

    Maud eCochet-Bissuel

    2014-05-01

    Full Text Available Ion channels are crucial components of cellular excitability and are involved in many neurological diseases. This review focuses on the sodium leak, G protein-coupled receptors (GPCRs-activated NALCN channel that is predominantly expressed in neurons where it regulates the resting membrane potential and neuronal excitability. NALCN is part of a complex that includes not only GPCRs, but also UNC-79, UNC-80, NLF-1 and src family of Tyrosine kinases (SFKs. There is growing evidence that the NALCN channelosome critically regulates its ion conduction. Both in mammals and invertebrates, animal models revealed an involvement in many processes such as locomotor behaviours, sensitivity to volatile anesthetics, and respiratory rhythms. There is also evidence that alteration in this NALCN channelosome can cause a wide variety of diseases. Indeed, mutations in the NALCN gene were identified in Infantile Neuroaxonal Dystrophy (INAD patients, as well as in patients with an Autosomal Recessive Syndrome with severe hypotonia, speech impairment, and cognitive delay. Deletions in NALCN gene were also reported in diseases such as 13q syndrome. In addition, genes encoding NALCN, NLF- 1, UNC-79 and UNC-80 proteins may be susceptibility loci for several diseases including bipolar disorder, schizophrenia, Alzheimer’s disease, autism, epilepsy, alcoholism, cardiac diseases and cancer. Although the physiological role of the NALCN channelosome is poorly understood, its involvement in human diseases should foster interest for drug development in the near future. Towards this goal, we review here the current knowledge on the NALCN channelosome in physiology and diseases.

  17. Sodium-level-sensitive sodium channel Na(x) is expressed in glial laminate processes in the sensory circumventricular organs.

    Science.gov (United States)

    Watanabe, Eiji; Hiyama, Takeshi Y; Shimizu, Hidetada; Kodama, Ryuji; Hayashi, Noriko; Miyata, Seiji; Yanagawa, Yuchio; Obata, Kunihiko; Noda, Masaharu

    2006-03-01

    Na(x) is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our recent studies on the Na(x)-gene-targeting mouse revealed that Na(x) channel is localized to the circumventricular organs (CVOs), the central loci for the salt and water homeostasis in mammals, where the Na(x) channel serves as a sodium-level sensor of the body fluid. To understand the cellular mechanism by which the information sensed by Na(x) channels is transferred to the activity of the organs, we dissected the subcellular localization of Na(x) in the present study. Double-immunostaining and immunoelectron microscopic analyses revealed that Na(x) is exclusively localized to perineuronal lamellate processes extended from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ, one of the CVOs, were sensitive to an increase in the extracellular sodium level, as analyzed by an ion-imaging method. These results suggest that glial cells bearing the Na(x) channel are the first to sense a physiological increase in the level of sodium in the body fluid, and they regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of the salt homeostasis.

  18. The voltage dependence of Ih in human myelinated axons

    Science.gov (United States)

    Howells, James; Trevillion, Louise; Bostock, Hugh; Burke, David

    2012-01-01

    HCN channels are responsible for Ih, a voltage-gated inwardly rectifying current activated by hyperpolarization. This current appears to be more active in human sensory axons than motor and may play a role in the determination of threshold. Differences in Ih are likely to be responsible for the high variability in accommodation to hyperpolarization seen in different subjects. The aim of this study was to characterise this current in human axons, both motor and sensory. Recordings of multiple axonal excitability properties were performed in 10 subjects, with a focus on the changes in threshold evoked by longer and stronger hyperpolarizing currents than normally studied. The findings confirm that accommodation to hyperpolarization is greater in sensory than motor axons in all subjects, but the variability between subjects was greater than the modality difference. An existing model of motor axons was modified to take into account the behaviour seen with longer and stronger hyperpolarization, and a mathematical model of human sensory axons was developed based on the data collected. The differences in behaviour of sensory and motor axons and the differences between different subjects are best explained by modulation of the voltage dependence, along with a modest increase of expression of the underlying conductance of Ih. Accommodation to hyperpolarization for the mean sensory data is fitted well with a value of −94.2 mV for the mid-point of activation (V0.5) of Ih as compared to −107.3 mV for the mean motor data. The variation in response to hyperpolarization between subjects is accounted for by varying this parameter for each modality (sensory: −89.2 to −104.2 mV; motor −87.3 to −127.3 mV). These voltage differences are within the range that has been described for physiological modulation of Ih function. The presence of slowly activated Ih isoforms on both motor and sensory axons was suggested by modelling a large internodal leak current and a masking of

  19. The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides.

    Science.gov (United States)

    Zhang, Yongqiang; Du, Yuzhe; Jiang, Dingxin; Behnke, Caitlyn; Nomura, Yoshiko; Zhorov, Boris S; Dong, Ke

    2016-09-16

    Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Inactivation properties of sodium channel Nav1.8 maintain action potential amplitude in small DRG neurons in the context of depolarization

    Directory of Open Access Journals (Sweden)

    Waxman Stephen G

    2007-05-01

    Full Text Available Abstract Background Small neurons of the dorsal root ganglion (DRG express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP. To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Nav1.8. Results In small neurons cultured from wild type (WT adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s sodium channels, and the tetrodotoxin-resistant (TTX-r Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/- mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/- DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties. Conclusion We conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.

  1. δ-opioid Receptor Induced Inhibition of Sodium Channel Function

    Institute of Scientific and Technical Information of China (English)

    康学智; 顾全保; 丁光宏; 晁东满; 王英伟; G Balboni; LH Lazarus; 夏萤

    2008-01-01

    Objective: To study the precise role of DOR in the regulation of sodium channels at present. Methods: With Xenopus oocytes co-expressing sodium channel subtype 2 (Nav1.2) and DOR. Results: 1) Nav1.2 expression induced tetrodotoxin-sensitive inward currents; 2) DOR expression reduced the inward currents; 3) activation of DOR reduced the amplitude of the current and rightly shifted the activation curve of the current in the oocytes with both Nav1.2 and DOR, but not in ones with Nav1.2 alone; 4) the DOR agonist-induced inhibition of Nav1.2 currents was in a dose-dependent manner and saturable; 5) the DOR agonist had no effect on naive oocytes. Conclusion: These data represent the first demonstration that activation of DOR inhibits Na+ channel function by decreasing the amplitude of sodium currents and increasing its threshold of activation. This novel finding has far-reaching impacts on novel solutions of certain neurological disorders such as hypoxic/ischemic injury, epilepsy and pain. Also, our data may improve the understanding of the mechanisms underlying acupuncture since acupuncture is known to activate the brain opioid system.%目的:研究δ-阿片受体表达和激活对钠通道1.2亚型的电流特性的影响.方法:用双电极电压钳技术,在δ-阿片受体和钠通道亚型1.2共表达的非洲爪蟾第V期卵母细胞上,观察δ-阿片受体表达和/或激活后,钠通道1.2亚型电流特性的变化.结果:1)钠通道1.2亚型的表达产生河豚毒素(tetrodotoxin,TTX)敏感的内向电流;2)δ-阿片受体的表达减少钠通道激活电流的幅度;3)δ-阿片受体和钠通道1.2亚型共表达的卵母细胞中,δ-阿片受体激动剂可以抑制钠通道激活电流的幅度和电导,而只有钠通道1.2亚型表达的卵母细胞则无此现象;4)δ-阿片受体激动剂抑制钠电流的作用具有剂量依赖关系,并能达到饱和状态;5)δ-阿片受体激动剂对未表达外派陛蛋白的卵母细胞无影响.结论:本结

  2. The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation

    DEFF Research Database (Denmark)

    Kaestner, Lars; Christophersen, Palle; Bernhardt, Ingolf;

    2000-01-01

    Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor......Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor...

  3. Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

    Science.gov (United States)

    Pinto, Bernardo I; García, Isaac E; Pupo, Amaury; Retamal, Mauricio A; Martínez, Agustín D; Latorre, Ramón; González, Carlos

    2016-07-22

    Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

  4. Could a defective epithelial sodium channel lead to bronchiectasis

    Directory of Open Access Journals (Sweden)

    Hubert Dominique

    2008-05-01

    Full Text Available Abstract Background Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis. Methods We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement, and 17 had no such evidence. Results Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E. The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport. Conclusion Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.

  5. Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

    Energy Technology Data Exchange (ETDEWEB)

    Ottea, J.A.; Payne, G.T.; Soderlund, D.M. (Cornell Univ., Geneva, NY (USA))

    1990-08-01

    Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealed that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.

  6. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    Science.gov (United States)

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  7. Sodium channel gene expression in mosquitoes, Aedes albopictus (S.)

    Institute of Scientific and Technical Information of China (English)

    NANNAN LIU; QIANG XU; LEE ZHANG

    2006-01-01

    A mosquito strain of Aerdes albopictus,HAmAalG0,from Huntsville,Alabama,USA,showed a normal susceptibility and low tolerance to permethrin and resmethrin (pyrethroid insecticides) compared to a susceptible Ikaken strain,even though these pyrethroid insecticides have been used in the field for a long period of time in Alabama.Recently,we treated HAmAalG0 in the laboratory with permethrin for five generations and detected no significant change in the level of resistance to permethrin in the selected mosquitoes,HAmAalG5,compared with the parental strain HAmAalG0. We then examined the allelic expression at the L-to-F kdr site of the sodium channel gene in the Aedes mosquitoes to address our hypothesis that the L-to-F kdr mutation was not present in HAmAalG0 and HAmAalG5 mosquitoes. We found that every tested individual in Ikaken,HAmAalG0,and HAmAalG5 populations expressed a codon of CTA at the L-to-F kdr site encoding Leu,strongly corresponding to their susceptibility to insecticides.

  8. The Sodium-Activated Potassium Channel Slack Is Required for Optimal Cognitive Flexibility in Mice

    Science.gov (United States)

    Bausch, Anne E.; Dieter, Rebekka; Nann, Yvette; Hausmann, Mario; Meyerdierks, Nora; Kaczmarek, Leonard K.; Ruth, Peter; Lukowski, Robert

    2015-01-01

    "Kcnt1" encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual…

  9. mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

    Science.gov (United States)

    Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

    1998-01-01

    It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

  10. Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches

    OpenAIRE

    1982-01-01

    We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel ...

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

    Science.gov (United States)

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

    2014-11-01

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

  12. Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation.

    Science.gov (United States)

    Fanger, G R; Vaillancourt, R R; Heasley, L E; Montmayeur, J P; Johnson, G L; Maue, R A

    1997-01-01

    The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.

  13. Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides.

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Luo, Ningguang; Liu, Zhiqi; Lee, Jung-Eun; Khambay, Bhupinder; Dong, Ke

    2009-01-15

    Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an alpha-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report the identification of a residue G(1111) and two positively charged lysines immediately downstream of G(1111) in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G(1111), a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G(1111) had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.

  14. Hapalindoles from the cyanobacterium fischerella: potential sodium channel modulators.

    Science.gov (United States)

    Cagide, Eva; Becher, Paul G; Louzao, M Carmen; Espiña, Begoña; Vieytes, Mercedes R; Jüttner, Friedrich; Botana, Luis M

    2014-10-20

    Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of (15)N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

  15. Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture.

    Science.gov (United States)

    Cohen, Lior; Ilan, Nitza; Gur, Maya; Stühmer, Walter; Gordon, Dalia; Gurevitz, Michael

    2007-10-05

    Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.

  16. Antiepileptic drugs targeting sodium channels: subunit and neuron-type specific interactions

    NARCIS (Netherlands)

    Qiao, X.

    2013-01-01

    Certain antiepileptic drugs (e.g. carbamazepine and lamotrigine) block sodium channels in an use-dependent manner and this mechanism contributes to the anti-convulsant properties of these drugs. There are, however, subtle differences in sodium current blocking properties of the antiepileptic drugs

  17. Antiepileptic drugs targeting sodium channels: subunit and neuron-type specific interactions

    NARCIS (Netherlands)

    Qiao, X.

    2013-01-01

    Certain antiepileptic drugs (e.g. carbamazepine and lamotrigine) block sodium channels in an use-dependent manner and this mechanism contributes to the anti-convulsant properties of these drugs. There are, however, subtle differences in sodium current blocking properties of the antiepileptic drugs w

  18. Regulatory role of voltage-gated sodium channel β subunits in sensory neurons

    Directory of Open Access Journals (Sweden)

    Mohamed eChahine

    2011-11-01

    Full Text Available Voltage-gated Na+ channels are transmembrane-bound proteins incorporating aqueous conduction pores that are highly selective for Na+. The opening of these channels results in the rapid influx of Na+ ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na+-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β1, β2 were first described. Subsequent cloning and heterologous expression studies revealed that the  subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, ten isoforms of the Na+ channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β1-β4 and two splice variants (β1A, β1B have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.

  19. Transient receptor potential vanilloid 1 activation by dietary capsaicin promotes urinary sodium excretion by inhibiting epithelial sodium channel α subunit-mediated sodium reabsorption.

    Science.gov (United States)

    Li, Li; Wang, Fei; Wei, Xing; Liang, Yi; Cui, Yuanting; Gao, Feng; Zhong, Jian; Pu, Yunfei; Zhao, Yu; Yan, Zhencheng; Arendshorst, William J; Nilius, Bernd; Chen, Jing; Liu, Daoyan; Zhu, Zhiming

    2014-08-01

    High salt (HS) intake contributes to the development of hypertension. Epithelial sodium channels play crucial roles in regulating renal sodium reabsorption and blood pressure. The renal transient receptor potential vanilloid 1 (TRPV1) cation channel can be activated by its agonist capsaicin. However, it is unknown whether dietary factors can act on urinary sodium excretion and renal epithelial sodium channel (ENaC) function. Here, we report that TRPV1 activation by dietary capsaicin increased urinary sodium excretion through reducing sodium reabsorption in wild-type (WT) mice on a HS diet but not in TRPV1(-/-) mice. The effect of capsaicin on urinary sodium excretion was involved in inhibiting αENaC and its related with-no-lysine kinase 1/serum- and glucocorticoid-inducible protein kinase 1 pathway in renal cortical collecting ducts of WT mice. Dietary capsaicin further reduced the increased αENaC activity in WT mice attributed to the HS diet. In contrast, this capsaicin effect was absent in TRPV1(-/-) mice. Immunoprecipitation study indicated αENaC specifically coexpressed and functionally interact with TRPV1 in renal cortical collecting ducts of WT mice. Additionally, ENaC activity and expression were suppressed by capsaicin-mediated TRPV1 activation in cultured M1-cortical collecting duct cells. Long-term dietary capsaicin prevented the development of high blood pressure in WT mice on a HS diet. It concludes that TRPV1 activation in the cortical collecting ducts by capsaicin increases urinary sodium excretion and avoids HS diet-induced hypertension through antagonizing αENaC-mediated urinary sodium reabsorption. Dietary capsaicin may represent a promising lifestyle intervention in populations exposed to a high dietary salt intake.

  20. [From gene to disease; primary erythermalgia--a neuropathic disease as a consequence of mutations in a sodium pump gene

    NARCIS (Netherlands)

    Drenth, J.P.H.; Morsche, R.H.M. te; Michiels, J.J.M.

    2006-01-01

    Primary erythermalgia is a rare autosomal dominant inherited disorder characterized by recurrent attacks of red, warm and painful burning extremities. The gene involved in primary erythermalgia, SCN9A, encodes for a voltage dependent sodium channel alpha subunit (NaV1.7). NaV1.7 is located in dorsal

  1. Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

    Energy Technology Data Exchange (ETDEWEB)

    Rosenberg, R.L.

    1985-01-01

    The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based on molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.

  2. Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches.

    Science.gov (United States)

    Patlak, J; Horn, R

    1982-03-01

    We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at -40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.

  3. Pattern of Functional TTX-Resistant Sodium Channels Reveals a Developmental Stage of Human iPSC- and ESC-Derived Nociceptors

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    Esther Eberhardt

    2015-09-01

    Full Text Available Human pluripotent stem cells (hPSCs offer the opportunity to generate neuronal cells, including nociceptors. Using a chemical-based approach, we generated nociceptive sensory neurons from HUES6 embryonic stem cells and retrovirally reprogrammed induced hPSCs derived from fibroblasts. The nociceptive neurons expressed respective markers and showed tetrodotoxin-sensitive (TTXs and -resistant (TTXr voltage-gated sodium currents in patch-clamp experiments. In contrast to their counterparts from rodent dorsal root ganglia, TTXr currents of hPSC-derived nociceptors unexpectedly displayed a significantly more hyperpolarized voltage dependence of activation and fast inactivation. This apparent discrepancy is most likely due to a substantial expression of the developmentally important sodium channel NAV1.5. In view of the obstacles to recapitulate neuropathic pain in animal models, our data advance hPSC-derived nociceptors as a better model to study developmental and pathogenetic processes in human nociceptive neurons and to develop more specific small molecules to attenuate pain.

  4. Voltage-dependent K+ currents contribute to heterogeneity of olfactory ensheathing cells

    Science.gov (United States)

    Rela, Lorena; Piantanida, Ana Paula; Bordey, Angelique; Greer, Charles A.

    2015-01-01

    The olfactory nerve is permissive for axon growth throughout life. This has been attributed in part to the olfactory ensheathing glial cells that encompass the olfactory sensory neuron fascicles. Olfactory ensheathing cells also promote axon growth in vitro and when transplanted in vivo to sites of injury. The mechanisms involved remain largely unidentified owing in part to the limited knowledge of the physiological properties of ensheathing cells. Glial cells rely for many functions on the properties of the potassium channels expressed; however, those expressed in ensheathing cells are unknown. Here we show that olfactory ensheathing cells express voltage-dependent potassium currents compatible with inward rectifier (Kir) and delayed rectifier (KDR) channels. Together with gap junction coupling, these contribute to the heterogeneity of membrane properties observed in olfactory ensheathing cells. The relevance of K+ currents expressed by ensheathing cells is discussed in relation to plasticity of the olfactory nerve. PMID:25856239

  5. Effects of BmKNJX11, a bioactive polypeptide purified from Buthus martensi Karsch, on sodium channels in rat dorsal root ganglion neurons.

    Science.gov (United States)

    Wang, Xi-Jie; An, Shan-Shan; Cheng, Hong; Xu, San-Hua; Cheng, Jie; Lu, Wei; Gao, Rong; Xiao, Hang

    2009-01-01

    A long-chain polypeptide BmKNJX11 was purified from the venom of Asian scorpion Buthus martensi Karsch (BmK) by a combination of gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. The molecular mass was found to be 7036.85 Da by electrospray ionization mass spectrometry. The first 15 N-terminal amino acid sequence of BmKNJX11 was determined to be GRDAY IADSE NCTYT by Edman degradation. With whole cell recording, BmKNJX11 inhibited tetrodotoxin-sensitive voltage-gated sodium channels (TTX-S VGSC) in freshly isolated rat dorsal root ganglion (DRG) neurons in a concentration- and voltage-dependent manner. At a concentration of 40 mug/ml BmKNJX11 lowered the activation threshold and produced negative shifting of TTX-S sodium current (I(Na)) activation curve. In addition, BmKNJX11 induced shifting of the steady-state inactivation curve to the left, delayed the recovery of TTX-S I(Na) from inactivation, and also reduced the fraction of available sodium channels. These results suggested that BmKNJX11 might exert effects on VGSC by binding to a specific site. Considering that TTX-S VGSC expressed in DRG neurons play a critical role in nociceptive transmission, the interaction of BmKNJX11 with TTX-S VGSC might lead to a change in excitability of nociceptive afferent fibers, which may be involved in the observed peripheral pain expression.

  6. Sodium ion channel mutations in glioblastoma patients correlate with shorter survival

    Directory of Open Access Journals (Sweden)

    Velculescu Victor E

    2011-02-01

    Full Text Available Abstract Background Glioblastoma Multiforme (GBM is the most common and invasive astrocytic tumor associated with dismal prognosis. Treatment for GBM patients has advanced, but the median survival remains a meager 15 months. In a recent study, 20,000 genes from 21 GBM patients were sequenced that identified frequent mutations in ion channel genes. The goal of this study was to determine whether ion channel mutations have a role in disease progression and whether molecular targeting of ion channels is a promising therapeutic strategy for GBM patients. Therefore, we compared GBM patient survival on the basis of presence or absence of mutations in calcium, potassium and sodium ion transport genes. Cardiac glycosides, known sodium channel inhibitors, were then tested for their ability to inhibit GBM cell proliferation. Results Nearly 90% of patients showed at least one mutation in ion transport genes. GBM patients with mutations in sodium channels showed a significantly shorter survival compared to patients with no sodium channel mutations, whereas a similar comparison based on mutational status of calcium or potassium ion channel mutations showed no survival differences. Experimentally, targeting GBM cells with cardiac glycosides such as digoxin and ouabain demonstrated preferential cytotoxicity against U-87 and D54 GBM cells compared to non-tumor astrocytes (NTAs. Conclusions These pilot studies of GBM patients with sodium channel mutations indicate an association with a more aggressive disease and significantly shorter survival. Moreover, inhibition of GBM cells by ion channel inhibitors such as cardiac glycosides suggest a therapeutic strategy with relatively safe drugs for targeting GBM ion channel mutations. Key Words: glioblastoma multiforme, ion channels, mutations, small molecule inhibitors, cardiac glycosides.

  7. Neurotoxins and their binding areas on voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Marijke eStevens

    2011-11-01

    Full Text Available Voltage-gated Sodium Channels (VGSCs are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Genetic defects in sodium channel genes therefore can cause a wide variety of diseases, generally called ‘channelopathies’.The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. Until now, these concepts still form the basis for understanding the functioning of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and will generate a massive influx of sodium ions. Immediately after, channels will start inactivating and currents decrease. In the inactivated state channels stay refractory for any new stimulus and they must return to the closed state before being susceptible to any new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX and saxitoxin (STX also contributed largely to our today’s understanding of the structure and function of ion channels and specifically of VGSCs. Moreover, neurotoxins acting on ion channels turned out to be valuable tools in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt®, Elan. The original peptide, -MVIIA, is derived from the cone snail Conus magus and now FDA/EMEA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in neurons.This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the

  8. Molecular Basis of Paraltyic Neurotoxin Action on Voltage-Sensitive Sodium Channels

    Science.gov (United States)

    1985-10-14

    of 9,700 daltons isolated from the coral Goni2oora gy. (1). The toxin enhances neurally mediated contraction of blood vessels and taenia coli of the...sites on the solium channel and to identify the site of GPT action within the structure of the sodium channel protein. 2. Site of Action of Brvyetoxin

  9. An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels.

    Science.gov (United States)

    Onwuli, Donatus O; Beltran-Alvarez, Pedro

    2016-03-01

    Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

  10. The Structural Basis and Functional Consequences of Interactions Between Tetrodotoxin and Voltage-Gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    C. Ruben

    2006-04-01

    Full Text Available Abstract: Tetrodotoxin (TTX is a highly specific blocker of voltage-gated sodium channels. The dissociation constant of block varies with different channel isoforms. Until recently, channel resistance was thought to be primarily imparted by amino acid substitutions at a single position in domain I. Recent work reveals a novel site for tetrodotoxin resistance in the P-region of domain IV.

  11. Manipulating the voltage dependence of tunneling spin torques

    KAUST Repository

    Manchon, Aurelien

    2012-10-01

    Voltage-driven spin transfer torques in magnetic tunnel junctions provide an outstanding tool to design advanced spin-based devices for memory and reprogrammable logic applications. The non-linear voltage dependence of the torque has a direct impact on current-driven magnetization dynamics and on devices performances. After a brief overview of the progress made to date in the theoretical description of the spin torque in tunnel junctions, I present different ways to alter and control the bias dependence of both components of the spin torque. Engineering the junction (barrier and electrodes) structural asymmetries or controlling the spin accumulation profile in the free layer offer promising tools to design effcient spin devices.

  12. Role of Sodium Channel on Cardiac Action Potential

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    S. H. Sabzpoushan

    2012-06-01

    Full Text Available Sudden cardiac death is a major cause of death worldwide. In most cases, it's caused by abnormal action potential propagation that leads to cardiac arrhythmia. The aim of this article is to study the abnormal action potential propagation through sodium ion concentration variations. We use a new electrophysiological model that is both detailed and computationally efficient. This efficient model is based on the partial differential equation method. The central finite difference method is used for numerical solving of the two-dimensional (2D wave propagation equation. Simulations are implemented in two stages, as a single cardiac cell and as a two-dimensional grid of cells. In both stages, the normal action potential formation in case of a single cell and it's normal propagation in case of a two-dimensional grid of cells were simulated with nominal sodium ion conductance. Then, the effect of sodium ion concentration on the action potential signal was studied by reducing the sodium ion conductance. It is concluded that reducing the sodium ion conductance, decreases both passing ability and conduction velocity of the action potential wave front.

  13. AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells.

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    Shaowu Xue

    Full Text Available The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na⁺ transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⁺ and K⁺ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⁺ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⁺ over K⁺ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⁺ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⁺ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⁺ channel transport properties.

  14. Sodium channel expression in the ventral posterolateral nucleus of the thalamus after peripheral nerve injury

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    Waxman Stephen G

    2006-08-01

    Full Text Available Abstract Peripheral nerve injury is known to up-regulate the expression of rapidly-repriming Nav1.3 sodium channel within first-order dorsal root ganglion neurons and second-order dorsal horn nociceptive neurons, but it is not known if pain-processing neurons higher along the neuraxis also undergo changes in sodium channel expression. In this study, we hypothesized that after peripheral nerve injury, third-order neurons in the ventral posterolateral (VPL nucleus of the thalamus undergo changes in expression of sodium channels. To test this hypothesis, adult male Sprague-Dawley rats underwent chronic constriction injury (CCI of the sciatic nerve. Ten days after CCI, when allodynia and hyperalgesia were evident, in situ hybridization and immunocytochemical analysis revealed up-regulation of Nav1.3 mRNA, but no changes in expression of Nav1.1, Nav1.2, or Nav1.6 in VPL neurons, and unit recordings demonstrated increased background firing, which persisted after spinal cord transection, and evoked hyperresponsiveness to peripheral stimuli. These results demonstrate that injury to the peripheral nervous system induces alterations in sodium channel expression within higher-order VPL neurons, and suggest that misexpression of the Nav1.3 sodium channel increases the excitability of VPL neurons injury, contributing to neuropathic pain.

  15. Nav1.7 is the predominant sodium channel in rodent olfactory sensory neurons

    Directory of Open Access Journals (Sweden)

    Black Joel A

    2011-05-01

    Full Text Available Abstract Background Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons. Methods We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs. Results We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system. Conclusions Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.

  16. Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development

    Directory of Open Access Journals (Sweden)

    Siguang Xu

    2016-01-01

    Full Text Available The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC and acid sensitive ionic channel (ASIC. ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na+ across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics.

  17. Genetic and environmental factors in cardiac sodium channel disease

    NARCIS (Netherlands)

    Mizusawa, Y.

    2016-01-01

    Cardiac sodium channelopathies, such as long QT syndrome type3 (LQT3), Brugada syndrome (BrS) and cardiac conduction disease (CCD), are heritable diseases associated with mutations in the SCN5A gene and sudden cardiac death. They were classically thought to be a monogenic disease. However, while LQT

  18. Genetic and environmental factors in cardiac sodium channel disease

    NARCIS (Netherlands)

    Mizusawa, Y.

    2016-01-01

    Cardiac sodium channelopathies, such as long QT syndrome type3 (LQT3), Brugada syndrome (BrS) and cardiac conduction disease (CCD), are heritable diseases associated with mutations in the SCN5A gene and sudden cardiac death. They were classically thought to be a monogenic disease. However, while LQT

  19. Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I).

    Science.gov (United States)

    Silva, Jonathan R; Goldstein, Steve A N

    2013-03-01

    In skeletal muscle, slow inactivation (SI) of Na(V)1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na(V)1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na(V)1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1-160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na(V)1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (~10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.

  20. Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I)

    Science.gov (United States)

    Silva, Jonathan R.

    2013-01-01

    In skeletal muscle, slow inactivation (SI) of NaV1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in NaV1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in NaV1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1–160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of NaV1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (∼10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX. PMID:23401572

  1. Effect of angiotensin II-induced arterial hypertension on the voltage-dependent contractions of mouse arteries.

    Science.gov (United States)

    Fransen, Paul; Van Hove, Cor E; Leloup, Arthur J A; Schrijvers, Dorien M; De Meyer, Guido R Y; De Keulenaer, Gilles W

    2016-02-01

    Arterial hypertension (AHT) affects the voltage dependency of L-type Ca(2+) channels in cardiomyocytes. We analyzed the effect of angiotensin II (AngII)-induced AHT on L-type Ca(2+) channel-mediated isometric contractions in conduit arteries. AHT was induced in C57Bl6 mice with AngII-filled osmotic mini-pumps (4 weeks). Normotensive mice treated with saline-filled osmotic mini-pumps were used for comparison. Voltage-dependent contractions mediated by L-type Ca(2+) channels were studied in vaso-reactive studies in vitro in isolated aortic and femoral arteries by using extracellular K(+) concentration-response (KDR) experiments. In aortic segments, AngII-induced AHT significantly sensitized isometric contractions induced by elevated extracellular K(+) and depolarization. This sensitization was partly prevented by normalizing blood pressure with hydralazine, suggesting that it was caused by AHT rather than by direct AngII effects on aortic smooth muscle cells. The EC50 for extracellular K(+) obtained in vitro correlated significantly with the rise in arterial blood pressure induced by AngII in vivo. The AHT-induced sensitization persisted when aortic segments were exposed to levcromakalim or to inhibitors of basal nitric oxide release. Consistent with these observations, AngII-treatment also sensitized the vaso-relaxing effects of the L-type Ca(2+) channel blocker diltiazem during K(+)-induced contractions. Unlike aorta, AngII-treatment desensitized the isometric contractions to depolarization in femoral arteries pointing to vascular bed specific responses of arteries to hypertension. AHT affects the voltage-dependent L-type Ca(2+) channel-mediated contraction of conduit arteries. This effect may contribute to the decreased vascular compliance in AHT and explain the efficacy of Ca(2+) channel blockers to reduce vascular stiffness and central blood pressure in AHT.

  2. Paramyotonia congenita and hyperkalemic periodic paralysis map to the same sodium-channel gene locus.

    Science.gov (United States)

    Ptacek, L J; Trimmer, J S; Agnew, W S; Roberts, J W; Petajan, J H; Leppert, M

    1991-01-01

    Paramyotonia congenita (PC), an autosomal dominant muscle disease, shares some clinical and electrophysiological similarities with another myotonic muscle disorder, hyperkalemic periodic paralysis (HYPP). However, clinical and electrophysiologic differences allow differentiation of the two disorders. The HYPP locus was recently shown to be linked to a skeletal muscle sodium-channel gene probe. We now report that PC maps to the same locus (LOD score 4.4, theta = 0 at assumed penetrance of .95). These linkage results, coupled with physiological data demonstrating abnormal sodium-channel function in patients with PC, implicate a sodium-channel gene as an important candidate for the site of mutation responsible for PC. Furthermore, this is strong evidence for the hypothesis that PC and HYPP are allelic disorders. PMID:1654742

  3. Use-dependent block of cardiac sodium channels by quaternary derivatives of lidocaine.

    Science.gov (United States)

    Gintant, G A; Hoffman, B F

    1984-02-01

    Modulation of the reduction of fast inward sodium current by local anesthetics due to changes in electrical activity has been termed use-dependent block ( Courtney 1975). To determine the mechanisms responsible for use-dependent block of cardiac sodium channels and to compare use-dependent block in cardiac and nerve preparations, we investigated use-dependent block of cardiac sodium channels by the quaternary lidocaine analogues QX -314 and QX -222 (two agents previously studied in nerve). We used canine cardiac Purkinje fibers, and assessed changes in the fast inward sodium current using changes in the maximum rate of rise of the action potential upstroke (Vmax). Two microelectrode voltage clamp and current clamp techniques were used to control membrane potential prior to stimulated upstrokes . Use-dependent block was not affected by shortening the action potential duration during rapid stimulation. Partial recovery from use-dependent block was observed during rapid stimulation with brief depolarizing prepulses terminating immediately prior to the upstroke. Similar prepulses also prevented the development of use-dependent block following an abrupt increase in the stimulation rate. Hyperpolarizing prepulses during rapid stimulation caused recovery from use-dependent block; recovery was greater and more rapid with increasingly negative prepulses . Hyperpolarization during periods of electrical quiescence also caused greater recovery. These results, interpreted using the modulated receptor hypothesis ( Hille 1977; Hondeghem and Katzung 1977), suggest that use-dependent block of cardiac sodium channels by quaternary local anesthetics is due to drug association with the inactivated sodium channel receptor which occurs only after these drugs gain access to the receptor site through open sodium channels.

  4. The study of sodium channels involved in pain responses using specific modulators

    Institute of Scientific and Technical Information of China (English)

    JI Yong-Hua; LIU Tong

    2008-01-01

    Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for generation and conduction of action potentials in excitable cells. Physiological and pharmacological studies have demonstrated that VGSCs play a critical role in chronic pain associated with tissue or nerve injury. Many long-chain peptide toxins (60-76 amino acid residues) purified from the venom of Asian scorpion Buthus martensii Karsch (BmK) axe investigated to be sodium channel-specific modulators. The α-like neurotoxins that can bind to receptor site 3 of sodium channels, named as BmK I and BmK abT, could induce nociceptive effects in rats. On the contrast, the β-like neurotoxins that can bind to receptor site 4 of sodium channels, named as BmK AS, BmK AS-1 and BmK IT2, could produce potent anti-nociceptive effects in animal pain models. BmK I could strongly prolong the fast inactivation of tetrodotoxin (TTX)- sensitive Na+ currents on the rat dorsal root ganglia (DRG) neurons together with the augmentation of peak current amplitude. Howevor BmK IT2 and BmK ASs, potently suppressed both the peak TTX-resistant and TTX-sensitive Na+ currents on rat small DRG neurons. Moreover, BmK ASs could decrease the excitability of small DRG neurons. Thus, the nociception/anti-nociception induced by scorpion neurotoxins may attribute to their distinct modulation on sodium channels in primary afferent sensory neurons. Therefore, the sodium channel-specific modulators from BmK venom could be used as not only pharmacological tools for better understanding the roles of VGSCs in pain signal conduction, but also lead molecules in the development of ideal analgesics targetingVGSCs.

  5. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    Science.gov (United States)

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  6. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  7. Thermal and chemical unfolding and refolding of a eukaryotic sodium channel.

    Science.gov (United States)

    Charalambous, Kalypso; O'Reilly, A O; Bullough, Per A; Wallace, B A

    2009-06-01

    Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with "denatured" channels, retaining significant helical secondary structure even at 77 degrees C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.

  8. Tetrahydroacridine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Wei WANG; Yi-ping WANG; Guo-yuan HU

    2004-01-01

    AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na+ channels in cardiac tissues.METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltagedependent Na+ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na+ current with an IC50 value of 120 μmol/L (95 % confidence range: 108-133 μmol/L). (2) The inhibitory effects of tacrine on Na+ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100 μmol/L caused a negative shift (about 10 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na+ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na+ channels in guinea-pig ventricular myocytes. Tactine acted as inactivation stabilizer of Na+channels in cardiac tissues.

  9. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  10. 增龄引起犬心房L型电压依赖型钙通道离子重构的分子机制%Aging-related ionic remodeling of L-type voltage dependent calcium channel in left atria of canine

    Institute of Scientific and Technical Information of China (English)

    周贤惠; 张健; 甘天翊; 许国军; 汤宝鹏

    2012-01-01

    目的 探讨增龄导致犬心房L型电压依赖型钙通道离子重构的分子机制.方法 采用全细胞膜片钳技术记录犬左心房肌细胞L型电压依赖型钙通道动作电位时限(APD90)、动作电位平台期电压和L型钙离子电流(ICa-L)特性.应用实时定量逆转录聚合酶链反应(RT-PCR)法测定犬左心耳L型电压依赖型钙通道α1亚单位(CaV1.2)、钙离子释放通道兰尼碱受体(RYR2)、肌浆网钙调控-Ca2+ ATP酶基因(SERCA2)、钙激活蛋白酶-Ⅰ(Calpain-Ⅰ)、磷酸受钠蛋白(PLN1)等的mRNA表达,用Western blot检测蛋白表达.结果 老年犬与成年犬比较,心房肌细胞L型电压依赖型钙通道APD90较长[(340.5±10.1)ms 比(320.0±7.9) ms,P<0.05];动作电位平台期电压较低[(-9.5±1.7)mV比(-6.4±1.1)mV,P<0.05];ICa-L电流密度较低[(-14.04±0.82)pA/pF比(-8.11±0.54)pA/pF,P<0.05].老年犬与成年犬比较,CaV1.2基因表达明显下调(0.90±0.35比2.38±0.40,P<0.05),RYR2基因表达明显上调(4.39±4.68比1.49±1.69,P<0.05),两组犬SERCA2、Calpain-Ⅰ、PLN1基因表达差异无统计学意义;CaV1.2蛋白表达明显下调(0.13±0.10比0.29±0.12,P<0.05),RYR2蛋白明显上调(0.18±0.21比0.08±0.36,P<0.05),两组犬SERCA2、Calpain-Ⅰ、PLN1蛋白表达无明显改变.结论 增龄导致犬心房肌细胞钙通道CaV1.2和RYR2基因和蛋白表达的改变是L型电压依赖型钙通道离子重构的分子机制,可能是老年相关性心房颤动的潜在机制之一.%Objective To investigate aging-related ionic remodeling of L-type voltage dependent calcium channel (LVDCC) in left atria of canine.Methods Seven adult (2.0-2.5 years ) and 10 aged (>8 years) dogs were used.The current of LVDCC was recorded by patch clamp technique in the whole cell mode.The action potential duration (APD90),amplitude of action potential plateau (APA),ICa-L peak current density of LVDCC were recorded. The mRNA and protein expressions of αlc subunit (CaV1

  11. Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Elstone, Fisal D; Niciforovic, Ana P

    2014-01-01

    functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect......Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain...... largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1...

  12. Studies of multimodal gating of the sodium channel.

    Science.gov (United States)

    Keynes, Richard D

    2002-01-01

    Chandler and Meves found that in squid axons perfused with NaF a small flow of Na+ ions persisted in the inactivated state, and that the Na+ channel therefore has more than one open state. Studies by Correa and Bezanilla on single patches in squid axons showed that such steady currents arose from reopening of the channel at a relatively low frequency. Currents with comparable properties are generated in mammalian brain cells and elsewhere. The existence of a third mode of gating was established by Patlak and Ortiz when they showed that in frog muscle fibres there were occasionally quite large bursts of late openings. Again, similar behaviour has been observed in other types of muscle and in brain cells. It is suggested that the voltage gating of all ionic channels involves a screw-helical mechanism, operating in steps each transferring unit charge. For segment S4 in domain IV of Na+ channels, three charges have to be transferred to reach the initial open state, and a fourth for fast inactivation to take place. The single late openings in the inactivated steady state may be explained by the transfer of a fifth charge in IVS4, while the larger bursts of reopening involve a modulation of the mechanism of fast inactivation.

  13. Molecular Model of Anticonvulsant Drug Binding to the Voltage-Gated Sodium Channel Inner Pore

    Science.gov (United States)

    Lipkind, Gregory M.

    2010-01-01

    The tricyclic anticonvulsant drugs phenytoin, carbamazepine, and lamotrigine block neuronal voltage-gated Na+ channels, and their binding sites to domain IV-S6 in the channel's inner pore overlap with those of local anesthetic drugs. These anticonvulsants are neutral, in contrast to the mostly positively charged local anesthetics, but their open/inactivated-state blocking affinities are similar. Using a model of the open pore of the Na+ channel that we developed by homology with the crystal structures of potassium channels, we have docked these three anticonvulsants with residues identified by mutagenesis as important for their binding energy. The three drugs show a common pharmacophore, including an aromatic ring that has an aromatic-aromatic interaction with Tyr-1771 of NaV1.2 and a polar amide or imide that interacts with the aromatic ring of Phe-1764 by a low-energy amino-aromatic hydrogen bond. The second aromatic ring is nearly at a right angle to the pharmacophore and fills the pore lumen, probably interacting with the other S6 segments and physically occluding the inner pore to block Na+ permeation. Hydrophobic interactions with this second aromatic ring may contribute an important component to binding for anticonvulsants, which compensates energetically for the absence of positive charge in their structures. Voltage dependence of block, their important therapeutic property, results from their interaction with Phe-1764, which connects them to the voltage sensors. Their use dependence is modest and this results from being neutral, with a fast drug off-rate after repolarization, allowing a normal action potential rate in the presence of the drugs. PMID:20643904

  14. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol

    OpenAIRE

    Patel, Reesha R.; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Cummins, Theodore R.

    2016-01-01

    Resurgent sodium currents arise from channel reopening during repolarisation, and are predicted to increase neuronal excitability. Patel et al. show that epilepsy-associated mutations in the voltage-gated sodium channel Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy.

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  16. Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use

    NARCIS (Netherlands)

    Waxman, S.G.; Merkies, I.S.; Gerrits, M.M.; Dib-Hajj, S.D.; Lauria, G.; Cox, J.J.; Wood, J.N.; Woods, C.G.; Drenth, J.P.H.; Faber, C.G.

    2014-01-01

    Human studies have firmly implicated voltage-gated sodium channels in human pain disorders, and targeted and massively parallel genomic sequencing is beginning to be used in clinical practice to determine which sodium channel variants are involved. Missense substitutions of SCN9A, the gene encoding

  17. Expression of mRNA coding voltage - gated sodium channel α-subunit in spontaneously epileptic rat

    Institute of Scientific and Technical Information of China (English)

    DUWa; CAIJi-Qun

    2004-01-01

    OBJECTIVE Subtypes Ⅰ,Ⅱ and Ⅲ of sodium channel α- subunit mRNA were analyzed in adult rat brain of spontaneously epileptic rats, and investigated the relationship between sodium channel expression and epilepsy. METHODS Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyms, observe

  18. Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus.

    NARCIS (Netherlands)

    Kortenoeven, M.L.A.; Li, Yuedan; Shaw, S.M.; Gaeggeler, H.P.; Rossier, B.C.; Wetzels, J.F.M.; Deen, P.M.T.

    2009-01-01

    Lithium therapy frequently induces nephrogenic diabetes insipidus; amiloride appears to prevent its occurrence in some clinical cases. Amiloride blocks the epithelial sodium channel (ENaC) located in the apical membrane of principal cells; hence one possibility is that ENaC is the main entry site fo

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  20. Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel

    NARCIS (Netherlands)

    Wilde, Arthur A. M.; Brugada, Ramon

    2011-01-01

    Variations in the gene encoding for the major sodium channel (Na(v)1.5) in the heart, SCN5A, has been shown to cause a number of arrhythmia syndromes (with or without structural changes in the myocardium), including the long-QT syndrome (type 3), Brugada syndrome, (progressive) cardiac conduction di

  1. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Morsche, R.H.M. te; Guillet, G.; Taieb, A.; Kirby, R.L.; Jansen, J.B.M.J.

    2005-01-01

    Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha

  2. Statistical analysis of single sodium channels. Effects of N-bromoacetamide.

    OpenAIRE

    Horn, R.; Vandenberg, C A; Lange, K.

    1984-01-01

    Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control an...

  3. Mutations in Sodium Channel Gene SCN9A and the Pain Perception Disorders

    OpenAIRE

    Marković, Danica; Janković, Radmilo; Veselinović, Ines

    2015-01-01

    Voltage-gated sodium channels (NaV) play a crucial role in development and propagation of action potentials in neurons and muscle cells. NaV1.7 channels take a special place in modern science since it is believed that they contribute to nerve hyperexcitability. Mutations of the gene SCN9A, which codes the α subunit of NaV1.7 channels, are associated with pain perception disorders (primary erythermalgia, congenital analgesia, and paroxysmal pain disorder). It is considered that the SCN9A gene ...

  4. Tetrodotoxin-sensitive α-subunits of voltage-gated sodium channels are relevant for inhibition of cardiac sodium currents by local anesthetics.

    Science.gov (United States)

    Stoetzer, C; Doll, T; Stueber, T; Herzog, C; Echtermeyer, F; Greulich, F; Rudat, C; Kispert, A; Wegner, F; Leffler, A

    2016-06-01

    The sodium channel α-subunit (Nav) Nav1.5 is regarded as the most prevalent cardiac sodium channel required for generation of action potentials in cardiomyocytes. Accordingly, Nav1.5 seems to be the main target molecule for local anesthetic (LA)-induced cardiotoxicity. However, recent reports demonstrated functional expression of several "neuronal" Nav's in cardiomyocytes being involved in cardiac contractility and rhythmogenesis. In this study, we examined the relevance of neuronal tetrodotoxin (TTX)-sensitive Nav's for inhibition of cardiac sodium channels by the cardiotoxic LAs ropivacaine and bupivacaine. Effects of LAs on recombinant Nav1.2, 1.3, 1.4, and 1.5 expressed in human embryonic kidney cell line 293 (HEK-293) cells, and on sodium currents in murine, cardiomyocytes were investigated by whole-cell patch clamp recordings. Expression analyses were performed by reverse transcription PCR (RT-PCR). Cultured cardiomyocytes from neonatal mice express messenger RNA (mRNA) for Nav1.2, 1.3, 1.5, 1.8, and 1.9 and generate TTX-sensitive sodium currents. Tonic and use-dependent block of sodium currents in cardiomyocytes by ropivacaine and bupivacaine were enhanced by 200 nM TTX. Inhibition of recombinant Nav1.5 channels was similar to that of TTX-resistant currents in cardiomyocytes but stronger as compared to inhibition of total sodium current in cardiomyocytes. Recombinant Nav1.2, 1.3, 1.4, and 1.5 channels displayed significant differences in regard to use-dependent block by ropivacaine. Finally, bupivacaine blocked sodium currents in cardiomyocytes as well as recombinant Nav1.5 currents significantly stronger in comparison to ropivacaine. Our data demonstrate for the first time that cardiac TTX-sensitive sodium channels are relevant for inhibition of cardiac sodium currents by LAs.

  5. Biophysical characterisation of the persistent sodium current of the Nav1.6 neuronal sodium channel: a single-channel analysis.

    Science.gov (United States)

    Chatelier, Aurélien; Zhao, Juan; Bois, Patrick; Chahine, Mohamed

    2010-06-01

    Na(v)1.6 is the major voltage-gated sodium channel at nodes of Ranvier. This channel has been shown to produce a robust persistent inward current in whole-cell experiments. Na(v)1.6 plays an important role in axonal conduction and may significantly contribute to the pathophysiology of the injured nervous system through this persistent current. However, the underlying molecular mechanisms and regulation of the persistent current are not well understood. Using the whole-cell configuration of the patch-clamp technique, we investigated the Na(v)1.6 transient and persistent currents in HEK-293. Previous studies have shown that the persistent current depended on the content of the patch electrode. Therefore, we characterised the single-channel properties of the persistent current with an intact intracellular medium using the cell-attached configuration of the patch-clamp technique. In HEK-293 cells, the Na(v)1.6 persistent current recorded in the whole-cell configuration was 3-5% of the peak transient current. In single-channel recording, the ratio between peak and persistent open probability confirmed the magnitude of the persistent current observed in the whole-cell configuration. The cell-attached configuration revealed that the molecular mechanism of the whole-cell persistent current is a consequence of single Na(v)1.6 channels reopening.

  6. Nav1.5 cardiac sodium channels, regulation and clinical implications

    Directory of Open Access Journals (Sweden)

    Henry Humberto León-Ariza

    2014-10-01

    Full Text Available Voltage-gated sodium channels constitute a group of membrane proteins widely distributed thought the body. In the heart, there are at least six different isoforms, being the Nav1.5 the most abundant. The channel is composed of an α subunit that is formed by four domains of six segments each, and four much smaller β subunits that provide stability and integrate other channels into the α subunit. The function of the Nav1.5 channel is modulated by intracellular cytoskeleton proteins, extracellular proteins, calcium concentration, free radicals, and medications, among other things. The study of the channel and its alterations has grown thanks to its association with pathogenic conditions such as Long QT syndrome, Brugada syndrome, atrial fibrillation, arrhythmogenic ventricular dysplasia and complications during ischemic processes.

  7. TPEN, a Specific Zn(2+) Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K(+) and Na(+) Channels.

    Science.gov (United States)

    Zhang, Feng; Ma, Xue-Ling; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Xie, Lai-Hua; Liu, Yan-Qiang

    2017-03-01

    Hypoxia-ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia-ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic-ischemic conditions. The effects of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn(2+)-chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-D-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K(+) and Na(+) channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I K as well as decreased GluR2 protein expression and Na(+) channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K(+) and Na(+) channels in neurons. Hence, Zn(2+) chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.

  8. The hitchhiker's guide to the voltage-gated sodium channel galaxy.

    Science.gov (United States)

    Ahern, Christopher A; Payandeh, Jian; Bosmans, Frank; Chanda, Baron

    2016-01-01

    Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure-function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na(+) selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts.

  9. Different effects of mexiletine on two mutant sodium channels causing paramyotonia congenita and hyperkalemic periodic paralysis.

    Science.gov (United States)

    Weckbecker, K; Würz, A; Mohammadi, B; Mansuroglu, T; George, A L; Lerche, H; Dengler, R; Lehmann-Horn, F; Mitrovic, N

    2000-01-01

    Effects of the antiarrhythmic and antimyotonic drug mexiletine were studied on two sodium channel mutants causing paramyotonia congenita (R1448H) and an overlap paramyotonic and hyperkalemic paralytic syndrome (M1360V). Channels were expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. Compared to the wild-type, channel, both mutants showed alterations of inactivation, i.e. slower inactivation, left shift of steady-state inactivation and faster recovery from inactivation. Mexiletine caused a significantly larger use-dependent block of the R1448H mutant when compared to M1360V and wild-type channels. This can be explained by a prolonged recovery from mexiletine block as observed for R1448H channels, since the affinity of mexiletine for the inactivated state was similar for all three clones. The use-dependent block of sodium channels by mexiletine reduces repetitive series of action potentials and therefore improves muscle stiffness in myotonic patients. The enhanced use-dependent block as seen with R1448H may explain the extraordinary therapeutic efficacy of mexiletine in most patients with paramyotonia congenita.

  10. Three Peptide Modulators of the Human Voltage-Gated Sodium Channel 1.7, an Important Analgesic Target, from the Venom of an Australian Tarantula

    Directory of Open Access Journals (Sweden)

    Chun Yuen Chow

    2015-06-01

    Full Text Available Voltage-gated sodium (NaV channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7. Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM, Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.

  11. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin

    DEFF Research Database (Denmark)

    Fuchs, W; Larsen, Erik Hviid; Lindemann, B

    1977-01-01

    ) was clamped to zero and step-changes of Na activity in the outer solution ((Na)(o)) were performed with a fast-flow chamber at constant ionic strength, while the short-circuit current was recorded.3. At pre-selected times after a step-change of (Na)(o) the current response (I) to a fast voltage staircase...... was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current-voltage curve of transmembrane and paracellular shunt pathways. The current-voltage curve of the Na channels was computed by subtracting the shunt current from the total current.4. The instantaneous I...... transport through open Na-selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)(o). However, when added to the outer border of the membrane Na causes a decrease of P(Na) within...

  12. Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges.

    Science.gov (United States)

    De Col, Roberto; Messlinger, Karl; Carr, Richard W

    2008-02-15

    Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na(+)-K(+)-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na(+)-K(+)-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10-300 microm) and carbamazepine (30-500 microm) but not tetrodotoxin (TTX, 10-80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na(+)-K(+)-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons.

  13. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides.

    Science.gov (United States)

    Silver, Kristopher S; Soderlund, David M

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel alpha subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na(v)1.2a, Na(v)1.4, Na(v)1.5, and Na(v)1.8 sodium channel alpha subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat beta1 auxiliary subunit on the sensitivity of the Na(v)1.2a and Na(v)1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel alpha subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na(v)1.4 > Na(v)1.2a > Na(v)1.5 > Na(v)1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na(v)1.8 isoform was most sensitive, the Na(v)1.4 isoform was completely insensitive, and the sensitivities of the Na(v)1.5 and Na(v)1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na(v)1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na(v)1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na(v)1.2a or Na(v)1.4 isoforms with the beta1 subunit was the modest reduction in the sensitivity of the Na(v)1.2a isoform to RH 3421. These results demonstrate that mammalian sodium channel isoforms differ in their sensitivities to pyrazoline-type insecticides.

  14. Voltage-dependent modulation of cardiac ryanodine receptors (RyR2 by protamine.

    Directory of Open Access Journals (Sweden)

    Paula L Diaz-Sylvester

    Full Text Available It has been reported that protamine (>10 microg/ml blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m; SR lumen-cytosol = 0 mV, protamine induced conductance transitions to several intermediate states (substates as well as full block of RyR2. At V(m>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin. A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

  15. Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act

    Science.gov (United States)

    Tucker, Kristal R.; Huertas, Marco A.; Horn, John P.; Canavier, Carmen C.; Levitan, Edwin S.

    2012-01-01

    Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment (AIS) and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency. PMID:23077037

  16. Scorpion beta-toxins and voltage-gated sodium channels: interactions and effects.

    Science.gov (United States)

    Pedraza Escalona, Martha; Possani, Lourival D

    2013-01-01

    Scorpion beta-toxins (beta-ScTxs) modify the activity of voltage-gated sodium (Nav) channels, thereby producing neurotoxic effects in diverse organisms. For this reason, beta-ScTxs are essential tools not only for discriminating among different channel sub-types but also for studying the mechanisms of channel gating and the structure-function relationship involved in this process. This review considers both the structural and the functional implications of the beta-ScTxs after they bind to their receptor sites, in accord with their classification into a) anti-mammalian beta-ScTxs, b) anti-insect selective excitatory beta-ScTxs, c) anti-insect selective depressant beta-ScTxs and d) beta-ScTxs active on both insect and mammals Nav channels. Additionally, the molecular mechanism of toxin action by the "voltage sensor trapping" model is discussed, and the systemic effects produced by these toxins are reviewed.

  17. Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine.

    Science.gov (United States)

    Dichgans, Martin; Freilinger, Tobias; Eckstein, Gertrud; Babini, Elena; Lorenz-Depiereux, Bettina; Biskup, Saskia; Ferrari, Michel D; Herzog, Jürgen; van den Maagdenberg, Arn M J M; Pusch, Michael; Strom, Tim M

    Familial hemiplegic migraine is an autosomal dominant severe subtype of migraine with aura characterised by some degree of hemiparesis during the attacks. So far, mutations in two genes regulating ion translocation-CACNA1A and ATP1A2-have been identified in pedigrees with this disease. To identify additional genes for familial hemiplegic migraine, we did a genome-wide linkage analysis of two disease pedigrees without mutations in CACNA1A and ATP1A2. Ion channel genes in the candidate interval were analysed for mutations, and the functional consequences of the recorded sequence alteration were determined. We identified a novel locus for familial hemiplegic migraine on chromosome 2q24. Sequencing of candidate genes in this region revealed a heterozygous missense mutation (Gln1489Lys) in the neuronal voltage-gated sodium channel gene SCN1A, mutations of which have been associated with epilepsy. This same mutation was present in three families with familial hemiplegic migraine. It results in a charge-altering aminoacid exchange in the so-called hinged-lid domain of the protein, which is critical for fast inactivation of the channel. Whole-cell recordings in transiently transfected tsA201 cells expressing the highly homologous SCN5A sodium channel showed that the mutation induces a two-fold to four-fold accelerated recovery from fast inactivation without altering any of the other channel parameters investigated. Dysfunction of the neuronal sodium channel SCN1A can cause familial hemiplegic migraine. Our findings have implications for the understanding of migraine aura. Moreover, our study reinforces the molecular links between migraine and epilepsy, two common paroxysmal disorders.

  18. Vestibular integrator neurons have quadratic functions due to voltage dependent conductances.

    Science.gov (United States)

    Magnani, Christophe; Eugène, Daniel; Idoux, Erwin; Moore, Lee E

    2013-12-01

    The nonlinear properties of the dendrites of the prepositus hypoglossi nucleus (PHN) neurons are essential for the operation of the vestibular neural integrator that converts a head velocity signal to one that controls eye position. A novel system of frequency probing, namely quadratic sinusoidal analysis (QSA), was used to decode the intrinsic nonlinear behavior of these neurons under voltage clamp conditions. Voltage clamp currents were measured at harmonic and interactive frequencies using specific nonoverlapping stimulation frequencies. Eigenanalysis of the QSA matrix reduces it to a remarkably compact processing unit, composed of just one or two dominant components (eigenvalues). The QSA matrix of rat PHN neurons provides signatures of the voltage dependent conductances for their particular dendritic and somatic distributions. An important part of the nonlinear response is due to the persistent sodium conductance (gNaP), which is likely to be essential for sustained effects needed for a neural integrator. It was found that responses in the range of 10 mV peak to peak could be well described by quadratic nonlinearities suggesting that effects of higher degree nonlinearities would add only marginal improvement. Therefore, the quadratic response is likely to sufficiently capture most of the nonlinear behavior of neuronal systems except for extremely large synaptic inputs. Thus, neurons have two distinct linear and quadratic functions, which shows that piecewise linear + quadratic analysis is much more complete than just piecewise linear analysis; in addition quadratic analysis can be done at a single holding potential. Furthermore, the nonlinear neuronal responses contain more frequencies over a wider frequency band than the input signal. As a consequence, they convert limited amplitude and bandwidth input signals to wider bandwidth and more complex output responses. Finally, simulations at subthreshold membrane potentials with realistic PHN neuron models

  19. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.

    Science.gov (United States)

    Drenth, Joost P H; te Morsche, Rene H M; Guillet, Gerard; Taieb, Alain; Kirby, R Lee; Jansen, Jan B M J

    2005-06-01

    Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha subunit SCN9A in patients with erythermalgia. The presence of voltage-gated sodium channels in sensory neurons is thought to play a crucial role in several chronic painful neuropathies. We examined four different families and two sporadic cases and detected missense sequence variants in SCN9A to be present in primary erythermalgia patients. A total of five of six mutations were located in highly conserved regions. One family with autosomal dominantly inherited erythermalgia was double heterozygous for two separate SCN9A mutations. These data establish primary erythermalgia as a neuropathic disorder and offers hope for treatment of this incapacitating painful disorder.

  20. Mutations at opposite ends of the DIII/S4-S5 linker of sodium channel NaV1.7 produce distinct pain disorders

    Directory of Open Access Journals (Sweden)

    Tyrrell Lynda

    2010-04-01

    Full Text Available Abstract Background Two groups of gain-of-function mutations in sodium channel NaV1.7, which are expressed in dorsal root ganglion (DRG neurons, produce two clinically-distinct pain syndromes - inherited erythromelalgia (IEM and paroxysmal extreme pain disorder (PEPD. IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of NaV1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of NaV1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers in the different domains (IEM: domains I and II; PEPD: domains III and IV, no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far. Results We have identified a new IEM mutation P1308L within the C-terminus of the DIII/S4-S5 linker of NaV1.7, ten amino acids from a known PEPD mutation V1298F which is located within the N-terminus of this linker. We used voltage-clamp to compare the biophysical properties of the two mutant channels and current-clamp to study their effects on DRG neuron excitability. We confirm that P1308L and V1298F behave as prototypical IEM and PEPD mutations, respectively. We also show that DRG neurons expressing either P1308L or V1298F become hyperexcitable, compared to DRG neurons expressing wild-type channels. Conclusions Our results provide evidence for differential roles of the DIII/S4-S5 linker N- and C-termini in channel

  1. Prevention of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels.

    Science.gov (United States)

    1992-02-10

    of a- scorpion toxins to sodium channels by interacting with the active site on the toxin . The effects of these reagents on the binding of a- scorpion ...whose effects did not reach statistical significance. For some peptides, the level of a- scorpion toxin binding was increased after preincubation with...developed a different assay to measure peptide effects on scorpion toxin binding. This assay took advantage of the CNaHA-1 cells which express Type hA

  2. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    OpenAIRE

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased a...

  3. Effect of Gamma Radiation on Sodium Channels in Different Conformations in Neuroblastoma Cells

    Science.gov (United States)

    1986-01-01

    rads). selectively sulphonic acid : Tris. 2-amino-2-hydroxymethylprepane-l.3- reduce sodium currents in isolated frog sciatic dial. nerves under...similar ful in demonstrating structural and functional spectrum of radiosensitivity. A large body of bio- properties of biological membrane macromole...channel function 12-41. One might expect that quences than does disruption of nucleic acids , ionizing radiation, comprising a higher energy spectrum than

  4. A novel anticonvulsant modulates voltage-gated sodium channel inactivation and prevents kindling-induced seizures.

    Science.gov (United States)

    Ashraf, Muhammad N; Gavrilovici, Cezar; Shah, Syed U Ali; Shaheen, Farzana; Choudhary, Muhammad I; Rahman, Atta-ur; Fahnestock, Margaret; Simjee, Shabana U; Poulter, Michael O

    2013-09-01

    Here, we explore the mechanism of action of isoxylitone (ISOX), a molecule discovered in the plant Delphinium denudatum, which has been shown to have anticonvulsant properties. Patch-clamp electrophysiology assayed the activity of ISOX on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy(kindling model). Quantitative transcription polymerase chain reaction (qRT-PCR) (QPCR) assessed brain-derived neurotrophic factor (BDNF) mRNA expression in kindled rats, and kindled rats treated with ISOX. ISOX suppressed sodium current (I(Na)) showing an IC50 value of 185 nM in cultured neurons. ISOX significantly slowed the recovery from inactivation (ISOX τ = 18.7 ms; Control τ = 9.4 ms; p kindled cortical neurons, the IC50 for sodium current block was identical to that found in cultured neurons. ISOX prevented kindled stage 5 seizures and decreased the enhanced BDNF mRNA expression that is normally associated with kindling (p kindling is likely a secondary outcome that nevertheless would suppress epileptogenesis. These data show a new class of anti-seizure compound that inhibits sodium channel function and prevents the development of epileptic seizures.

  5. Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Worley, P.F.; Baraban, J.M.

    1987-05-01

    The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using (/sup 3/H)batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of (/sup 3/H)BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of (/sup 3/H)BTX-B binding sites in forebrain sections also agrees with biochemical determinations. Autoradiographic localizations indicate that (/sup 3/H)BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations.

  6. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    Science.gov (United States)

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

  7. Designing a C84 fullerene as a specific voltage-gated sodium channel blocker

    Science.gov (United States)

    Hilder, Tamsyn A.; Chung, Shin-Ho

    2013-07-01

    Fullerene derivatives demonstrate considerable potential for numerous biological applications, such as the effective inhibition of HIV protease. Recently, they were identified for their ability to indiscriminately block biological ion channels. A fullerene derivative which specifically blocks a particular ion channel could lead to a new set of drug leads for the treatment of various ion channel-related diseases. Here, we demonstrate their extraordinary potential by designing a fullerene which mimics some of the functions of μ-conotoxin, a peptide derived from cone snail venom which potently binds to the bacterial voltage-gated sodium channel (NavAb). We show, using molecular dynamics simulations, that the C84 fullerene with six lysine derivatives uniformly attached to its surface is selective to NavAb over a voltage-gated potassium channel (Kv1.3). The side chain of one of the lysine residues protrudes into the selectivity filter of the channel, while the methionine residues located just outside of the channel form hydrophobic contacts with the carbon atoms of the fullerene. The modified C84 fullerene strongly binds to the NavAb channel with an affinity of 46 nM but binds weakly to Kv1.3 with an affinity of 3 mM. This potent blocker of NavAb may serve as a structural template from which potent compounds can be designed for the targeting of mammalian Nav channels. There is a genuine need to target mammalian Nav channels as a form of treatment of various diseases which have been linked to their malfunction, such as epilepsy and chronic pain.

  8. Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel alpha subunit.

    Science.gov (United States)

    Cantrell, A R; Smith, R D; Goldin, A L; Scheuer, T; Catterall, W A

    1997-10-01

    Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI5-24. cBIMPS had similar effects on type IIA brain Na+ channel alpha subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na+ channel alpha subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (LI-II) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in LI-II of the Na+ channel alpha subunit, causing a reduction in Na+ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.

  9. Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

    Directory of Open Access Journals (Sweden)

    Stefan Walzik

    Full Text Available Alternative splicing creates several Na(v1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v1.5 splice variants have been discovered. Four of them, namely Na(v1.5a, Na(v1.5c, Na(v1.5d, and Na(v1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3 and isolated cardiac conduction disease (CCD. When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v1.5, hNa(v1.5a, and hNa(v1.5c. In contrast, the hNa(v1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v1.5e, the neonatal variant of hNa(v1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

  10. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    Science.gov (United States)

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  11. Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

    Science.gov (United States)

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A; Gordon, Dalia; Gurevitz, Michael

    2010-10-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward

  12. Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family

    CERN Document Server

    Kaufman, I; Tindjong, R; McClintock, P V E; Eisenberg, R S

    2013-01-01

    We use Brownian dynamics simulations to study the permeation properties of a generic electrostatic model of a biological ion channel as a function of the fixed charge Q_f at its selectivity filter. We reconcile the recently-discovered discrete calcium conduction bands M0 (Q_f=1e), M1 (3e), M2 (5e) with the set of sodium conduction bands L0 (0.5-0.7e), L1 (1.5-2e) thereby obtaining a completed pattern of conduction and selectivity bands v Q_f for the sodium-calcium channels family. An increase of Q_f leads to an increase of calcium selectivity: L0 (sodium selective, non-blocking channel) -> M0 (non-selective channel) -> L1 (sodium selective channel with divalent block) -> M1 (calcium selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L1 band is identified with the eukaryotic (DEKA) sodium channel, and L0 (speculatively) with the bacterial NaChBac channel. The scheme created is able to account for the experimentally observed mutation-induced ...

  13. The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    Science.gov (United States)

    Valkova, Christina; Liebmann, Lutz; Krämer, Andreas; Hübner, Christian A.; Kaether, Christoph

    2017-01-01

    Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs. PMID:28117367

  14. Functional characterization of two novel scorpion sodium channel toxins from Lychas mucronatus.

    Science.gov (United States)

    Xu, Lingna; Li, Tian; Liu, Honglian; Yang, Fan; Liang, Songping; Cao, Zhijian; Li, Wenxin; Wu, Yingliang

    2014-11-01

    The diverse α-scorpion toxins are invaluable pharmacological tools and potential drugs targeting sodium channels, but the pharmacological profiles of most toxins remains unknown so far. Here, we reported pharmacological activities of two novel α-scorpion toxins LmαTX3 and LmαTX5 from the Lychas mucronatus. Using the expression vector pET-28a, the recombinant LmαTX3 and LmαTX5 were separated by RP-HPLC and identified by MALDI-TOF-MS. Subsequently, sodium channels rNav1.2, mNav1.4, hNav1.5 and hNav1.7 were used for evaluating the pharmacological activities of LmαTX3 and LmαTX5 toxins. The electrophysiological experiments showed that both 10 μM recombinant LmαTX3 and LmαTX5 seriously inhibited the fast inactivation of mNav1.4 and hNav1.5 channels, moderately affected hNav1.7 channel, and hardly modulated rNav1.2 channel. The dose-response experiments further indicated the EC50 values of LmαTX3 were 4.98 ± 0.79 μM for mNav1.4, 1.23 ± 0.31 μM for hNav1.5 and 31.46 ± 2.32 μM for hNav1.7 channels, respectively. Similar pharmacological profiles of recombinant LmαTX5 were also observed, and its EC50 values were 4.53 ± 1.38 μM, 1.03 ± 0.43 μM and 67.62 ± 2.31 μM for mNav1.4, hNav1.5 and hNav1.7, respectively. In addition, the recombinant LmαTX3 from the vector pET-14b had much less effect on the fast inactivation of mNav1.4, hNav1.5 and hNav1.7 channels, which indicated that the expression vector pET-14b likely played a critical role in toxin function. Together, these findings first highlighted that scorpion toxins from L. mucronatus were a new molecular resource of discovering pharmacological probes and prospective drugs targeting sodium channels in the future.

  15. Energetics of ion competition in the DEKA selectivity filter of neuronal sodium channels

    Directory of Open Access Journals (Sweden)

    D. Boda

    2015-03-01

    Full Text Available The energetics of ionic selectivity in the neuronal sodium channels is studied. A simple model constructed for the selectivity filter of the channel is used. The selectivity filter of this channel type contains aspartate (D, glutamate (E, lysine (K, and alanine (A residues (the DEKA locus. We use Grand Canonical Monte Carlo simulations to compute equilibrium binding selectivity in the selectivity filter and to obtain various terms of the excess chemical potential from a particle insertion procedure based on Widom's method. We show that K+ ions in competition with Na+ are efficiently excluded from the selectivity filter due to entropic hard sphere exclusion. The dielectric constant of protein has no effect on this selectivity. Ca2+ ions, on the other hand, are excluded from the filter due to a free energetic penalty which is enhanced by the low dielectric constant of protein.

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

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

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

  17. Interaction between the sodium channel inactivation linker and domain III S4-S5.

    Science.gov (United States)

    Smith, M R; Goldin, A L

    1997-10-01

    The III-IV linker (L(III-IV)) of the rat brain sodium channel is critical for fast inactivation, possibly forming a fast inactivation particle. Inactivation can be disrupted by mutation of a conserved alanine at position 1329 in the S4-S5 loop of domain III. Combination of a charged mutation at 1329 with a compensatory (opposite) charge mutation at position 1489 in L(III-IV) partially restores inactivation of the channel. The compensatory charge mutant channel has a single-channel mean open time that is similar to that of the wild-type channel and is approximately 50 times shorter than that of the L(III-IV) mutant channel. The results of thermodynamic cycle analysis indicate that the mutations in domain III S4-S5 and L(III-IV) have a coupling energy of 2.8 kcal/mol, indicating that the two mutations act interdependently. These data suggest that L(III-IV) interacts directly with A1329, which may form part of the docking site if L(III-IV) is a fast inactivation particle.

  18. TGF-β1, released by myofibroblasts, differentially regulates transcription and function of sodium and potassium channels in adult rat ventricular myocytes.

    Directory of Open Access Journals (Sweden)

    Kuljeet Kaur

    Full Text Available Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (I(Na density at -40 mV than myocytes in control medium (p3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both I(Na and I(to. In current-clamp experiments TGF-β1 (10 ng/ml prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05; at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased I(Na density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I(Na density was -36.25±2.8 pA/pF in control, -59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01, and -58.22±6.6 pA/pF at 1 ng/ml (p<0.01. In sharp contrast, I(to density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001 at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (Na(V1.5 (+73%; p<0.01, while reducing KCNIP2 (Kchip2; -77%; p<0.01 and KCND2 (K(V4.2; -50% p<0.05 mRNA levels. Further, the TGF-β1-induced increase in I(Na was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A. TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury.

  19. TGF-β1, Released by Myofibroblasts, Differentially Regulates Transcription and Function of Sodium and Potassium Channels in Adult Rat Ventricular Myocytes

    Science.gov (United States)

    Kaur, Kuljeet; Zarzoso, Manuel; Ponce-Balbuena, Daniela; Guerrero-Serna, Guadalupe; Hou, Luqia; Musa, Hassan; Jalife, José

    2013-01-01

    Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (INa) density at −40 mV than myocytes in control medium (p3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both INa and Ito. In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased INa density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. INa density was −36.25±2.8 pA/pF in control, −59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and −58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, Ito density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (NaV1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; −77%; p<0.01) and KCND2 (KV4.2; −50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in INa was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury. PMID:23393573

  20. Role of Ca(2+) in injury-induced changes in sodium current in rat skeletal muscle.

    Science.gov (United States)

    Filatov, Gregory N; Pinter, Martin J; Rich, Mark M

    2009-08-01

    Characteristics of voltage-dependent sodium current recorded from adult rat muscle fibers in loose patch mode were rapidly altered following nearby impalement with a microelectrode. Hyperpolarized shifts in the voltage dependence of activation and fast inactivation occurred within minutes. In addition, the amplitude of the maximal sodium current decreased within 30 min of impalement. Impalement triggered a sustained elevation of intracellular Ca(2+). However, buffering Ca(2+) by loading fibers with AM-BAPTA did not affect the hyperpolarized shifts in activation and inactivation, although it did prevent the reduction in current amplitude. Surprisingly, the rise in intracellular Ca(2+) occurred even in the absence of extracellular Ca(2+). This result indicated that the injury-induced Ca(2+) increase came from an intracellular source, but it was not blocked by an inhibitor of release from the sarcoplasmic reticulum, which suggested involvement of mitochondria. Ca(2+) release from mitochondria triggered by carbonyl cyanide 3-chlorophenylhydrazone was sufficient to cause a reduction in sodium current amplitude but had little effect of the voltage dependence of activation and fast inactivation. Our data suggest the effects of muscle injury can be separated into a Ca(2+)-dependent reduction in amplitude and a largely Ca(2+)-independent shift in activation and fast inactivation. Together, the impalement-induced changes in sodium current reduce the number of sodium channels available to open at the resting potential and may limit further depolarization and thus promote survival of muscle fibers following injury.

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

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

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

  2. Analytical Model for Voltage-Dependent Photo and Dark Currents in Bulk Heterojunction Organic Solar Cells

    OpenAIRE

    2016-01-01

    A physics-based explicit mathematical model for the external voltage-dependent forward dark current in bulk heterojunction (BHJ) organic solar cells is developed by considering Shockley-Read-Hall (SRH) recombination and solving the continuity equations for both electrons and holes. An analytical model for the external voltage-dependent photocurrent in BHJ organic solar cells is also proposed by incorporating exponential photon absorption, dissociation efficiency of bound electron-hole pairs (...

  3. Imidazol-1-ylethylindazole Voltage-Gated Sodium Channel Ligands Are Neuroprotective during Optic Neuritis in a Mouse Model of Multiple Sclerosis

    Science.gov (United States)

    2014-01-01

    A series of imidazol-1-ylethylindazole sodium channel ligands were developed and optimized for sodium channel inhibition and in vitro neuroprotective activity. The molecules exhibited displacement of a radiolabeled sodium channel ligand and selectivity for blockade of the inactivated state of cloned neuronal Nav channels. Metabolically stable analogue 6 was able to protect retinal ganglion cells during optic neuritis in a mouse model of multiple sclerosis. PMID:24601592

  4. Down regulation of sodium channels in the central nervous system of hibernating snails.

    Science.gov (United States)

    Kiss, T; Battonyai, I; Pirger, Z

    2014-05-28

    Hibernation, as behavior, is an evolutionary mode of adaptation of animal species to unfavorable environmental conditions. It is generally characterized by suppressed metabolism, which also includes down regulation of the energy consuming ion-channel functioning. Experimental data regarding decreased ion-channel function are scarce. Therefore, our goal was to study the possible down regulation of voltage-gated sodium channel (NaV) subtypes in the neurons of hibernating snails. Our immunohistochemical experiments revealed that the expression of NaV1.8-like channels in the central nervous system was substantially down regulated in hibernating animals. In contrast to NaV1.8-like, the NaV1.9-like channels were present in neurons independently from hibernating and non-hibernating states. Our western blot data supported the immunohistochemical results according to which the band of the NaV1.8-like channel protein was less intensively labeled in the homogenate of the hibernating snails. The NaV1.9-like immunoreactivity was equally present both in hibernating and active snails. Micro-electrophysiological experiments show that in hibernating snails both NaV1.8- and NaV1.9-like currents are substantially decreased compared to that of the active snails. The contradictory electrophysiological and immunohistochemical or western blot data suggest that the molecular mechanisms of the "channel arrest" could be different in diverse NaV channel subtypes. Climate changes will affect temperature extremes and a question is how different species beyond their physiological tolerance will or able to adapt to changing environment. Hibernation is an important mode of adaptation to extreme climatic variations, and pursuant to this the present results may contribute to the study of the behavioral ecology.

  5. A voltage-dependent persistent sodium current in mammalian hippocampal neurons

    OpenAIRE

    1990-01-01

    Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight- seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These curren...

  6. Kinetic changes and modulation by carbamazepine on voltage-gated sodium channels in rat CA1 neurons after epilepsy.

    NARCIS (Netherlands)

    G. Sun; T.R. Werkman; W.J. Wadman

    2006-01-01

    AIM: To study whether the functional properties of sodium channels, and subsequently the channel modulation by carbamazepine (CBZ) in hippocampal CA1 neurons can be changed after epileptic seizures. METHODS: We used the acutely dissociated hippocampal CA1 pyramidal cells from epilepsy model rats 3 w

  7. Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

    Science.gov (United States)

    DeCaen, Paul G; Takahashi, Yuka; Krulwich, Terry A; Ito, Masahiro; Clapham, David E

    2014-11-11

    Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

  8. Leaky sodium channels from voltage sensor mutations in periodic paralysis, but not paramyotonia

    Science.gov (United States)

    Francis, David G.; Rybalchenko, Volodymyr; Struyk, Arie

    2011-01-01

    Background: Hypokalemic periodic paralysis (HypoPP) is associated with mutations in either the CaV1.1 calcium channel or the NaV1.4 sodium channel. Some NaV1.4 HypoPP mutations have been shown to cause an anomalous inward current that may contribute to the attacks of paralysis. Herein, we test whether disease-associated NaV1.4 mutations in previously untested homologous regions of the channel also give rise to the anomalous current. Methods: The functional properties of mutant NaV1.4 channels were studied with voltage-clamp techniques in an oocyte expression system. Results: The HypoPP mutation NaV1.4-R1132Q conducts an anomalous gating pore current, but the homologous R1448C mutation in paramyotonia congenita does not. Conclusions: Gating pore currents arising from missense mutations at arginine residues in the voltage sensor domains of NaV1.4 are a common feature of HypoPP mutant channels and contribute to the attacks of paralysis. PMID:21490317

  9. Increased renal sodium absorption by inhibition of prostaglandin synthesis during fasting in healthy man. A possible role of the epithelial sodium channels

    Directory of Open Access Journals (Sweden)

    Graffe Carolina C

    2010-10-01

    Full Text Available Abstract Background Treatment with prostaglandin inhibitors can reduce renal function and impair renal water and sodium excretion. We tested the hypotheses that a reduction in prostaglandin synthesis by ibuprofen treatment during fasting decreased renal water and sodium excretion by increased absorption of water and sodium via the aquaporin2 water channels and the epithelial sodium channels. Methods The effect of ibuprofen, 600 mg thrice daily, was measured during fasting in a randomized, placebo-controlled, double-blinded crossover study of 17 healthy humans. The subjects received a standardized diet on day 1, fasted at day 2, and received an IV infusion of 3% NaCl on day 3. The effect variables were urinary excretions of aquaporin2 (u-AQP2, the beta-fraction of the epithelial sodium channel (u-ENaCbeta, cyclic-AMP (u-cAMP, prostaglandin E2 (u-PGE2. Free water clearance (CH2O, fractional excretion of sodium (FENa, and plasma concentrations of vasopressin, angiotensin II, aldosterone, atrial-, and brain natriuretic peptide. Results Ibuprofen decreased u-AQP2, u-PGE2, and FENa at all parts of the study. During the same time, ibuprofen significantly increased u-ENaCbeta. Ibuprofen did not change the response in p-AVP, u-c-AMP, urinary output, and free water clearance during any of these periods. Atrial-and brain natriuretic peptide were higher. Conclusion During inhibition of prostaglandin synthesis, urinary sodium excretion decreased in parallel with an increase in sodium absorption and increase in u-ENaCbeta. U-AQP2 decreased indicating that water transport via AQP2 fell. The vasopressin-c-AMP-axis did not mediate this effect, but it may be a consequence of the changes in the natriuretic peptide system and/or the angiotensin-aldosterone system Trial Registration Clinical Trials Identifier: NCT00281762

  10. Clopidogrel attenuates lithium-induced alterations in renal water and sodium channels/transporters in mice.

    Science.gov (United States)

    Zhang, Yue; Peti-Peterdi, János; Heiney, Kristina M; Riquier-Brison, Anne; Carlson, Noel G; Müller, Christa E; Ecelbarger, Carolyn M; Kishore, Bellamkonda K

    2015-12-01

    Lithium (Li) administration causes deranged expression and function of renal aquaporins and sodium channels/transporters resulting in nephrogenic diabetes insipidus (NDI). Extracellular nucleotides (ATP/ADP/UTP), via P2 receptors, regulate these transport functions. We tested whether clopidogrel bisulfate (CLPD), an antagonist of ADP-activated P2Y(12) receptor, would affect Li-induced alterations in renal aquaporins and sodium channels/transporters. Adult mice were treated for 14 days with CLPD and/or Li and euthanized. Urine and kidneys were collected for analysis. When administered with Li, CLPD ameliorated polyuria, attenuated the rise in urine prostaglandin E2 (PGE2), and resulted in significantly higher urinary arginine vasopressin (AVP) and aldosterone levels as compared to Li treatment alone. However, urine sodium excretion remained elevated. Semi-quantitative immunoblotting revealed that CLPD alone increased renal aquaporin 2 (AQP2), Na-K-2Cl cotransporter (NKCC2), Na-Cl cotransporter (NCC), and the subunits of the epithelial Na channel (ENaC) in medulla by 25-130 %. When combined with Li, CLPD prevented downregulation of AQP2, Na-K-ATPase, and NKCC2 but was less effective against downregulation of cortical α- or γ-ENaC (70 kDa band). Thus, CLPD primarily attenuated Li-induced downregulation of proteins involved in water conservation (AVP-sensitive), with modest effects on aldosterone-sensitive proteins potentially explaining sustained natriuresis. Confocal immunofluorescence microscopy revealed strong labeling for P2Y(12)-R in proximal tubule brush border and blood vessels in the cortex and less intense labeling in medullary thick ascending limb and the collecting ducts. Therefore, there is the potential for CLPD to be directly acting at the tubule sites to mediate these effects. In conclusion, P2Y(12)-R may represent a novel therapeutic target for Li-induced NDI.

  11. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    Science.gov (United States)

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  12. Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7

    Directory of Open Access Journals (Sweden)

    Rush Anthony M

    2007-01-01

    Full Text Available Abstract Background The disabling chronic pain syndrome erythromelalgia (also termed erythermalgia is characterized by attacks of burning pain in the extremities induced by warmth. Pharmacological treatment is often ineffective, but the pain can be alleviated by cooling of the limbs. Inherited erythromelalgia has recently been linked to mutations in the gene SCN9A, which encodes the voltage-gated sodium channel Nav1.7. Nav1.7 is preferentially expressed in most nociceptive DRG neurons and in sympathetic ganglion neurons. It has recently been shown that several disease-causing erythromelalgia mutations alter channel-gating behavior in a manner that increases DRG neuron excitability. Results Here we tested the effects of temperature on gating properties of wild type Nav1.7 and mutant L858F channels. Whole-cell voltage-clamp measurements on wild type or L858F channels expressed in HEK293 cells revealed that cooling decreases current density, slows deactivation and increases ramp currents for both mutant and wild type channels. However, cooling differentially shifts the midpoint of steady-state activation in a depolarizing direction for L858F but not for wild type channels. Conclusion The cooling-dependent shift of the activation midpoint of L858F to more positive potentials brings the threshold of activation of the mutant channels closer to that of wild type Nav1.7 at lower temperatures, and is likely to contribute to the alleviation of painful symptoms upon cooling in affected limbs in patients with this erythromelalgia mutation.

  13. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states.

    Science.gov (United States)

    Payandeh, Jian; Gamal El-Din, Tamer M; Scheuer, Todd; Zheng, Ning; Catterall, William A

    2012-05-20

    In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

  14. Analysis in a large hyperkalemic periodic paralysis pedigree supports tight linkage to a sodium channel locus.

    Science.gov (United States)

    Ptacek, L J; Tyler, F; Trimmer, J S; Agnew, W S; Leppert, M

    1991-01-01

    Hyperkalemic periodic paralysis (HYPP) is an autosomal dominant muscle disease with electrophysiological abnormalities suggesting a defect in a voltage-gated sodium channel (NaCh) gene. A human NaCh gene was recently shown to cosegregate with the disease allele in a family with HYPP. Using an independent clone, we have demonstrated close genetic linkage between an NaCh gene and the HYPP locus in another family. With physiological data demonstrating abnormal NaCh function in HYPP patients, the absence of any obligate recombinations in the two families strengthens the argument that this NaCh gene is the site of the defect in this disorder. PMID:1651050

  15. Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations.

    Science.gov (United States)

    Hawkins, Nicole A; Kearney, Jennifer A

    2016-01-01

    Mutations in voltage-gated sodium channel genes cause several types of human epilepsies. Often, individuals with the same sodium channel mutation exhibit diverse phenotypes. This suggests that factors beyond the primary mutation influence disease severity, including genetic modifiers. Mouse epilepsy models with voltage-gated sodium channel mutations exhibit strain-dependent phenotype variability, supporting a contribution of genetic modifiers in epilepsy. The Scn2a(Q54) (Q54) mouse model has a strain-dependent epilepsy phenotype. Q54 mice on the C57BL/6J (B6) strain exhibit delayed seizure onset and improved survival compared to [B6xSJL/J]F1.Q54 mice. We previously mapped two dominant modifier loci that influence Q54 seizure susceptibility and identified Hlf (hepatic leukemia factor) as a candidate modifier gene at one locus. Hlf and other PAR bZIP transcription factors had previously been associated with spontaneous seizures in mice thought to be caused by down-regulation of the pyridoxine pathway. An Hlf targeted knockout mouse model was used to evaluate the effect of Hlf deletion on Q54 phenotype severity. Hlf(KO/KO);Q54 double mutant mice exhibited elevated frequency and reduced survival compared to Q54 controls. To determine if direct modulation of the pyridoxine pathway could alter the Q54 phenotype, mice were maintained on a pyridoxine-deficient diet for 6 weeks. Dietary pyridoxine deficiency resulted in elevated seizure frequency and decreased survival in Q54 mice compared to control diet. To determine if Hlf could modify other epilepsies, Hlf(KO/+) mice were crossed with the Scn1a(KO/+) Dravet syndrome mouse model to examine the effect on premature lethality. Hlf(KO/+);Scn1a(KO/+) offspring exhibited decreased survival compared to Scn1a(KO/+) controls. Together these results demonstrate that Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations and that modulation of the pyridoxine pathway can also influence phenotype

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  17. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate

    OpenAIRE

    Ivanov, Vadim; Ivanova, Svetlana; KALINOVSKY, TATIANA; NIEDZWIECKI, ALEKSANDRA; Rath, Matthias

    2016-01-01

    Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition...

  18. Voltage-gated sodium channels: pharmaceutical targets via anticonvulsants to treat epileptic syndromes.

    Science.gov (United States)

    Abdelsayed, Mena; Sokolov, Stanislav

    2013-01-01

    Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel's fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.

  19. Molecular insights into the local anesthetic receptor within voltage-gated sodium channels using hydroxylated analogues of mexiletine

    Directory of Open Access Journals (Sweden)

    Jean-François eDesaphy

    2012-02-01

    Full Text Available We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003, suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogues by adding one or two hydroxyl groups to the aryl moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration-response relationships were constructed using an holding potential of -120 mV at 0.1 Hz (tonic block and 10 Hz (use-dependent block stimulation frequencies. The half-maximum inhibitory concentrations (IC50 were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. Alteration of tonic block suggests that the aryl moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryl moiety may modify high-affinity binding of the drug ammine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis showing that the presence of hydroxyl groups to the aryl moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with

  20. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.

    Science.gov (United States)

    Senatore, Adriano; Guan, Wendy; Boone, Adrienne N; Spafford, J David

    2014-04-25

    T-type (Cav3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCav3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Cav3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Nav1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.

  1. Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons: inhibition by BDS-I toxin.

    Science.gov (United States)

    Martina, Marco; Metz, Alexia E; Bean, Bruce P

    2007-01-01

    We characterized the kinetics and pharmacological properties of voltage-activated potassium currents in rat cerebellar Purkinje neurons using recordings from nucleated patches, which allowed high resolution of activation and deactivation kinetics. Activation was exceptionally rapid, with 10-90% activation in about 400 mus at +30 mV, near the peak of the spike. Deactivation was also extremely rapid, with a decay time constant of about 300 mus near -80 mV. These rapid activation and deactivation kinetics are consistent with mediation by Kv3-family channels but are even faster than reported for Kv3-family channels in other neurons. The peptide toxin BDS-I had very little blocking effect on potassium currents elicited by 100-ms depolarizing steps, but the potassium current evoked by action potential waveforms was inhibited nearly completely. The mechanism of inhibition by BDS-I involves slowing of activation rather than total channel block, consistent with the effects described in cloned Kv3-family channels and this explains the dramatically different effects on currents evoked by short spikes versus voltage steps. As predicted from this mechanism, the effects of toxin on spike width were relatively modest (broadening by roughly 25%). These results show that BDS-I-sensitive channels with ultrafast activation and deactivation kinetics carry virtually all of the voltage-dependent potassium current underlying repolarization during normal Purkinje cell spikes.

  2. Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

    Institute of Scientific and Technical Information of China (English)

    Xu Zhang; Mengdie Xia; Yang Li; Huihui Liu; Xin Jiang; Wenlin Ren; Jianping Wu

    2013-01-01

    NaChBac is a bacterial voltage-gated sodium (Nav) channel that shows sequence similarity to voltage-gated calcium channels.To understand the ion-permeation mechanism of Nav channels,we combined molecular dynamics simulation,structural biology and electrophysiological approaches to investigate the recently determined structure of NavRh,a marine bacterial NaChBac ortholog.Two Na+ binding sites are identified in the selectivity filter (SF) in our simulations:The extracellular Na+ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183,and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyi oxygens of Leu179 and Thr178.In contrast,Ca2+ ions are prone to being trapped by Glu183 at site 1,which then blocks the entrance of both Na+ and Ca2+ to the vestibule of the SF.In addition,Na+ permeates through the selective filter in an asymmetrical manner,a feature that resembles that of the mammalian Nav orthologs.The study reported here provides insights into the mechanism of ion selectivity on Na+ over Ca2+ in mammalian Nav channels.

  3. A sodium channel gene SCN9A polymorphism that increases nociceptor excitability.

    Science.gov (United States)

    Estacion, Mark; Harty, T Patrick; Choi, Jin-Sung; Tyrrell, Lynda; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2009-12-01

    Sodium channel Na(V)1.7, encoded by the SCN9A gene, is preferentially expressed in nociceptive primary sensory neurons, where it amplifies small depolarizations. In studies on a family with inherited erythromelalgia associated with Na(V)1.7 gain-of-function mutation A863P, we identified a nonsynonymous single-nucleotide polymorphism within SCN9A in the affected proband and several unaffected family members; this polymorphism (c. 3448C&T, Single Nucleotide Polymorphisms database rs6746030, which produces the amino acid substitution R1150W in human Na(V)1.7 [hNa(V)1.7]) is present in 1.1 to 12.7% of control chromosomes, depending on ethnicity. In this study, we examined the effect of the R1150W substitution on function of the hNa(V)1.7 channel, and on the firing of dorsal root ganglion (DRG) neurons in which this channel is normally expressed. We show that this polymorphism depolarizes activation (7.9-11mV in different assays). Current-clamp analysis shows that the 1150W allele depolarizes (6mV) resting membrane potential and increases ( approximately 2-fold) the firing frequency in response to depolarization in DRG neurons in which it is present. Our results suggest that polymorphisms in the Na(V)1.7 channel may influence susceptibility to pain.

  4. Mechanism of Ion Permeation in Mammalian Voltage-Gated Sodium Channels.

    Directory of Open Access Journals (Sweden)

    Somayeh Mahdavi

    Full Text Available Recent determination of the crystal structures of bacterial voltage-gated sodium (NaV channels have raised hopes that modeling of the mammalian counterparts could soon be achieved. However, there are substantial differences between the pore domains of the bacterial and mammalian NaV channels, which necessitates careful validation of mammalian homology models constructed from the bacterial NaV structures. Such a validated homology model for the NaV1.4 channel was constructed recently using the extensive mutagenesis data available for binding of μ-conotoxins. Here we use this NaV1.4 model to study the ion permeation mechanism in mammalian NaV channels. Linking of the DEKA residues in the selectivity filter with residues in the neighboring domains is found to be important for keeping the permeation pathway open. Molecular dynamics simulations and potential of mean force calculations reveal that there is a binding site for a Na+ ion just inside the DEKA locus, and 1-2 Na+ ions can occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting that inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The NaV1.4 model also provides a good description of the observed Na+/K+ selectivity.

  5. Compound-Specific Effects of Mutations at Val787 in DII-S6 of Nav1.4 Sodium Channels on the Action of Sodium Channel Inhibitor Insecticides

    Science.gov (United States)

    von Stein, Richard T.; Soderlund, David M.

    2012-01-01

    Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119

  6. Chloride channels mediate sodium sulphide-induced relaxation in rat uteri.

    Science.gov (United States)

    Mijušković, Ana; Kokić, Aleksandra Nikolić; Dušić, Zorana Oreščanin; Slavić, Marija; Spasić, Mihajlo B; Blagojević, Duško

    2015-07-01

    Hydrogen sulphide reduces uterine contractility and is of potential interest as a treatment for uterine disorders. The aim of this study was to explore the mechanism of sodium sulphide (Na2 S)-induced relaxation of rat uterus, investigate the importance of redox effects and ion channel-mediated mechanisms, and any interactions between these two mechanisms. Organ bath studies were employed to assess the pharmacological effects of Na2 S in uterine strips by exposing them to Na2 S with or without Cl(-) channel blockers (DIDS, NFA, IAA-94, T16Ainh-A01, TA), raised KCl (15 and 75 mM), K(+) channel inhibitors (glibenclamide, TEA, 4-AP), L-type Ca(2+) channel activator (S-Bay K 8644), propranolol and methylene blue. The activities of antioxidant enzymes were measured in homogenates of treated uteri. The expression of bestrophin channel 1 (BEST-1) was determined by Western blotting and RT-PCR. Na2 S caused concentration-dependent reversible relaxation of spontaneously active and calcium-treated uteri, affecting both amplitude and frequency of contractions. Uteri exposed to 75 mM KCl were less sensitive to Na2 S compared with uteri in 15 mM KCl. Na2 S-induced relaxations were abolished by DIDS, but unaffected by other modulators or by the absence of extracellular HCO3 (-) , suggesting the involvement of chloride ion channels. Na2 S in combination with different modulators provoked specific changes in the anti-oxidant profiles of uteri. The expression of BEST-1, both mRNA and protein, was demonstrated in rat uteri. The relaxant effects of Na2 S in rat uteri are mediated mainly via a DIDS-sensitive Cl(-) -pathway. Components of the relaxation are redox- and Ca(2+) -dependent. © 2015 The British Pharmacological Society.

  7. Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

    Science.gov (United States)

    Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

    2016-01-01

    Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

  8. Expression patterns, mutation detection and RNA interference of Rhopalosiphum padi voltage-gated sodium channel genes

    Science.gov (United States)

    Zuo, Yayun; Peng, Xiong; Wang, Kang; Lin, Fangfei; Li, Yuting; Chen, Maohua

    2016-07-01

    The voltage-gated sodium channel (VGSC) is the target of sodium-channel-blocking insecticides. Traditionally, animals were thought to have only one VGSC gene comprising a α-subunit with four homologous domains (DI-DIV). The present study showed that Rhopalosiphum padi, an economically important crop pest, owned a unique heterodimeric VGSC (H1 and H2 subunits) encoded by two genes (Rpvgsc1 and Rpvgsc2), which is unusual in insects and other animals. The open reading frame (ORF) of Rpvgsc1 consisted 1150 amino acids, and the ORF of Rpvgsc2 had 957 amino acids. Rpvgsc1 showed 64.1% amino acid identity to DI-DII of Drosophila melanogaster VGSC and Rpvgsc2 showed 64.0% amino acid identity to DIII-DIV of D. melanogaster VGSC. A M918L mutation previously reported in pyrethroids-resistant strains of other insects was found in the IIS4-S6 region of R. padi field sample. The two R. padi VGSC genes were expressed at all developmental stages and showed similar expression patterns after treatment with beta-cypermethrin. Knockdown of Rpvgsc1 or Rpvgsc2 caused significant reduction in mortality rate of R. padi after exposure to beta-cypermethrin. These findings suggest that the two R. padi VGSC genes are both functional genes.

  9. Predictably Convergent Evolution of Sodium Channels in the Arms Race between Predators and Prey.

    Science.gov (United States)

    Brodie, Edmund D; Brodie, Edmund D

    2015-09-01

    Evolution typically arrives at convergent phenotypic solutions to common challenges of natural selection. However, diverse molecular and physiological mechanisms may generate phenotypes that appear similar at the organismal level. How predictable are the molecular mechanisms of adaptation that underlie adaptive convergence? Interactions between toxic prey and their predators provide an excellent avenue to investigate the question of predictability because both taxa must adapt to the presence of defensive poisons. The evolution of resistance to tetrodotoxin (TTX), which binds to and blocks voltage-gated sodium channels (NaV1) in nerves and muscle, has been remarkably parallel across deep phylogenetic divides. In both predators and prey, representing three major vertebrate groups, TTX resistance has arisen through structural changes in NaV1 proteins. Fish, amphibians and reptiles, though they differ in the total number of NaV1 paralogs in their genomes, have each evolved common amino acid substitutions in the orthologous skeletal muscle NaV1.4. Many of these substitutions involve not only the same positions in the protein, but also the identical amino acid residues. Similarly, predictable convergence is observed across the family of sodium channel genes expressed in different tissues in puffer fish and in garter snakes. Trade-offs between the fundamental role of NaV1 proteins in selective permeability of Na+ and their ability to resist binding by TTX generate a highly constrained adaptive landscape at the level of the protein. © 2015 S. Karger AG, Basel.

  10. Relationship between action potential sodium channels and muscarinic receptors in mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Mack, J.E.

    1986-01-01

    Cholinergic agonists and antagonists were tested for their ability to influence stimulated and unstimulated /sup 22/Na uptake in preparations of forebrain and hindbrain in mice in vitro. In mouse forebrain, atropine and pirenzepine decreased stimulated sodium uptake. Dicyclomine decreased stimulated uptake in both the forebrain and hindbrain. McN-A-343 decreased stimulated sodium uptake in the forebrain. The effects of sodium channel ligands on muscarinic receptors was investigated in forebrain and hindbrain preparations. In the forebrain, veratridine and aconitine appeared to inhibit the binding of (/sup 3/H)QNB in a competitive manner. Tetrodotoxin alone had not effect on binding, but enhanced the inhibition by veratridine, with no effect on aconitine inhibition. In the hindbrain, veratridine appeared to inhibit (/sup 3/H)QNB binding non-competitively and competitively. The addition of magnesium increased the K/sub i/ value in the veratridine inhibition. GTP enhanced the inhibition by veratridine. Tetrodotoxin increased the K/sub i/ value of the veratridine inhibition curve. Tetrodotoxin alone also inhibited (/sup 3/H)QNB binding. Tetrodotoxin inhibited QNB binding in both a non-competitive and uncompetitive manner.

  11. Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations

    Directory of Open Access Journals (Sweden)

    Juang Jyh-Ming

    2009-02-01

    Full Text Available Abstract The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM. Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively, and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV. In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.

  12. Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

    Directory of Open Access Journals (Sweden)

    Anna Keppner

    Full Text Available The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC. To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD, was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

  13. Vector spin modeling for magnetic tunnel junctions with voltage dependent effects

    Energy Technology Data Exchange (ETDEWEB)

    Manipatruni, Sasikanth, E-mail: sasikanth.manipatruni@intel.com; Nikonov, Dmitri E.; Young, Ian A. [Exploratory Integrated Circuits, Components Research, Intel Corp., Hillsboro, Oregon 97124 (United States)

    2014-05-07

    Integration and co-design of CMOS and spin transfer devices requires accurate vector spin conduction modeling of magnetic tunnel junction (MTJ) devices. A physically realistic model of the MTJ should comprehend the spin torque dynamics of nanomagnet interacting with an injected vector spin current and the voltage dependent spin torque. Vector spin modeling allows for calculation of 3 component spin currents and potentials along with the charge currents/potentials in non-collinear magnetic systems. Here, we show 4-component vector spin conduction modeling of magnetic tunnel junction devices coupled with spin transfer torque in the nanomagnet. Nanomagnet dynamics, voltage dependent spin transport, and thermal noise are comprehended in a self-consistent fashion. We show comparison of the model with experimental magnetoresistance (MR) of MTJs and voltage degradation of MR with voltage. Proposed model enables MTJ circuit design that comprehends voltage dependent spin torque effects, switching error rates, spin degradation, and back hopping effects.

  14. Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

    Science.gov (United States)

    Martin, Lewis J; Corry, Ben

    2014-07-01

    Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

  15. Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

    Directory of Open Access Journals (Sweden)

    Lewis J Martin

    2014-07-01

    Full Text Available Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

  16. The Snake with the Scorpion's Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus).

    Science.gov (United States)

    Yang, Daryl C; Deuis, Jennifer R; Dashevsky, Daniel; Dobson, James; Jackson, Timothy N W; Brust, Andreas; Xie, Bing; Koludarov, Ivan; Debono, Jordan; Hendrikx, Iwan; Hodgson, Wayne C; Josh, Peter; Nouwens, Amanda; Baillie, Gregory J; Bruxner, Timothy J C; Alewood, Paul F; Lim, Kelvin Kok Peng; Frank, Nathaniel; Vetter, Irina; Fry, Bryan G

    2016-10-18

    Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous

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

    Directory of Open Access Journals (Sweden)

    Yiangou Yiangos

    2010-06-01

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

  18. A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

    Directory of Open Access Journals (Sweden)

    Li Jia

    2011-11-01

    Full Text Available Abstract Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa channels, which suggests that ion channel regulatory partners have evolved distinct lineage

  19. Role of the epithelial sodium channel in salt-sensitive hypertension

    Institute of Scientific and Technical Information of China (English)

    Yan SUN; Jia-ning ZHANG; Dan ZHAO; Qiu-shi WANG; Yu-chun GU; He-ping MA; Zhi-ren ZHANG

    2011-01-01

    The epithelial sodium channel (ENaC) is a heteromeric channel composed of three similar but distinct subunits, a, β and Y. This channel is an end-effector in the rennin-angiotensin-aldosterone system and resides in the apical plasma membrane of the renal cortical collecting ducts, where reabsorption of Na+ through ENaC is the final renal adjustment step for Na+ balance. Because of its regulation and function, the ENaC plays a critical role in modulating the homeostasis of Na+ and thus chronic blood pressure. The development of most forms of hypertension requires an increase in Na+ and water retention. The role of ENaC in developing high blood pressure is exemplified in the gain-of-function mutations in ENaC that cause Liddle's syndrome, a severe but rare form of inheritable hypertension.The evidence obtained from studies using animal models and in human patients indicates that improper Na+ retention by the kidney elevates blood pressure and induces salt-sensitive hypertension.

  20. Negative-dominance phenomenon with genetic variants of the cardiac sodium channel Nav1.5.

    Science.gov (United States)

    Sottas, Valentin; Abriel, Hugues

    2016-07-01

    During the past two decades, many pathological genetic variants in SCN5A, the gene encoding the pore-forming subunit of the cardiac (monomeric) sodium channel Na(v)1.5, have been described. Negative dominance is a classical genetic concept involving a "poison" mutant peptide that negatively interferes with the co-expressed wild-type protein, thus reducing its cellular function. This phenomenon has been described for genetic variants of multimeric K(+) channels, which mechanisms are well understood. Unexpectedly, several pathologic SCN5A variants that are linked to Brugada syndrome also demonstrate such a dominant-negative (DN) effect. The molecular determinants of these observations, however, are not yet elucidated. This review article summarizes recent findings that describe the mechanisms underlying the DN phenomenon of genetic variants of K(+), Ca(2+), Cl(-) and Na(+) channels, and in particular Brugada syndrome variants of Na(v)1.5. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  1. Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.

    Science.gov (United States)

    Böhm, J; Scherzer, S; Shabala, S; Krol, E; Neher, E; Mueller, T D; Hedrich, R

    2016-03-07

    The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. A novel mutation in the sodium channel α1 subunit gene in a child with Dravet syndrome in Turkey

    Institute of Scientific and Technical Information of China (English)

    Mutluay Arslan; Ulu(c) Yi(s); Hande (C)a(g)layan; R1dvan Akin

    2013-01-01

    Dravet syndrome is a rare epileptic encephalopathy characterized by frequent seizures beginning in the first year of life and behavioral disorders. Mutations in the sodium channel α1 subunit gene are the main cause of this disease. We report two patients with refractory seizures and psychomotor retardation in whom the final diagnosis was Dravet syndrome with confirmed mutations in the sodium channel α1 subunit gene. The mutation identified in the second patient was a novel frame shift mutation, which resulted from the deletion of five nucleotides in exon 24.

  3. Actions of Tefluthrin on Rat Nav1.7 Voltage-Gated Sodium Channels Expressed in Xenopus Oocytes

    OpenAIRE

    Tan, Jianguo; Soderlund, David M.

    2011-01-01

    In rats expression of the Nav1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Nav1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 µM modified of Nav1.7 chan...

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

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

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

  5. Neuropathic pain in two-generation twins carrying the sodium channel Nav1.7 functional variant R1150W.

    Science.gov (United States)

    Harrer, Judith U; Uçeyler, Nurcan; Doppler, Kathrin; Fischer, Tanya Z; Dib-Hajj, Sulayman D; Waxman, Stephen G; Sommer, Claudia

    2014-10-01

    We present clinical, neuropathological, and molecular genetic findings of a family with a new pain phenotype of the sodium channel gene SCN9A polymorphism R1150W. A 46-year-old woman presented with a 5-year history of episodic temperature- and exercise-dependent burning pain of the feet and lower legs associated with numbness of the distal upper and lower limbs. Her monozygotic twin sister and their mother and her twin presented similar symptoms. Clinical evaluation was normal except for a mild distal sensory deficit in fingers and feet. Electrophysiological testing was unremarkable, as were serum and cerebrospinal fluid laboratory findings. Skin biopsies of the distal lower limbs revealed an epidermal nerve fiber density at the lower limit of normal. Myelinated dermal nerve fibers showed elongated nodes of Ranvier, but normal distribution of nodal and paranodal proteins. Genetic testing for ion channel-associated pain disorders revealed an amino acid R1150W substitution of the Nav1.7 sodium channel. The combination of a Nav1.7 polymorphism with dysmyelinating features in small-caliber peripheral nerves has not been described before and may suggest an explanation for the clinical syndrome in our patients. Treatment with the sodium channel blocker lamotrigine provided some relief, consistent with a role of sodium channel dysfunction in the pain syndrome of this family.

  6. Voltage-dependent currents in microvillar receptor cells of the frog vomeronasal organ.

    Science.gov (United States)

    Trotier, D; Døving, K B; Rosin, J F

    1993-08-01

    Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 microns) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was -61 +/- 13 mV (mean +/- SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of -60 to -86 mV, with almost no spontaneous action potential. The input resistance was in the G omega range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2-10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near -45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near -75 mV. An outward K+ current was blocked by internal Cs+ ions or by external tetraethylammonium or Ba2+ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd2+ ions. A voltage-dependent Ca2+ current was observed in an iso-osmotic BaCl2 solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.

  7. Malignant hyperthermia-like reaction in a family with a sodium channel mutation at residue 1306

    Energy Technology Data Exchange (ETDEWEB)

    Vita, G.M.; Jedlicka, A.E.; Levitt, R.C. [Johns Hopkins Medical Institute, Baltimore, MD (United States)] [and others

    1994-09-01

    Malignant hyperthermia susceptibility (MHS) is an autosomal dominant, hypermetabolic disorder, triggered by potent inhalational anesthetics. We have previously suggeste the skeletal muscle sodium channel {alpha}-subunit (SCN4A) as a gene candidate to explain some forms of MHS. To evaluate this gene for mutations that might lead to a MHS-like episode, we amplified genomic DNA by PCR and used SSCP to screen each exon. We studied multiple MHS families which may be linked to this gene. The proband and a sibling from one of these families suspected of having MHS experienced trismus and body rigidity after induction of anesthesia. The caffiene and halothane contracture test proved diagnostic in these individuals and EMG studies suggested a form of myotomia. A mutation co-segregating with the myotonia/MHS phenotype was found in the region of exon 22.

  8. Pathophysiology and treatment of resistant hypertension: the role of aldosterone and amiloride-sensitive sodium channels.

    Science.gov (United States)

    Judd, Eric K; Calhoun, David A; Warnock, David G

    2014-01-01

    Resistant hypertension is a clinically distinct subgroup of hypertension defined by the failure to achieve blood pressure control on optimal dosing of at least 3 antihypertensive medications of different classes, including a diuretic. The pathophysiology of hypertension can be attributed to aldosterone excess in more than 20% of patients with resistant hypertension. Existing dogma attributes the increase in blood pressure seen with increases in aldosterone to its antinatriuretic effects in the distal nephron. However, emerging research, which has identified and has begun to define the function of amiloride-sensitive sodium channels and mineralocorticoid receptors in the systemic vasculature, challenges impaired natriuresis as the sole cause of aldosterone-mediated resistant hypertension. This review integrates these findings to better define the role of the vasculature and aldosterone in the pathophysiology of resistant hypertension. In addition, a brief guide to the treatment of resistant hypertension is presented.

  9. Proteolytic activation of the epithelial sodium channel ENaC in preeclampsia examined with urinary exosomes

    DEFF Research Database (Denmark)

    Nielsen, Maria Ravn; Rytz, Mie; Frederiksen-Møller, Britta

    2015-01-01

    OBJECTIVES: Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity...... as a positive control for the presence of collecting duct membrane. RESULTS: Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group (p... pregnancy and preeclampsia CONCLUSIONS: It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ-ENaC, 2) Urine exosome fraction displays a variable pattern of γ-ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women...

  10. Multiple mutations and mutation combinations in the sodium channel of permethrin resistant mosquitoes, Culex quinquefasciatus

    Science.gov (United States)

    Li, Ting; Zhang, Lee; Reid, William R.; Xu, Qiang; Dong, Ke; Liu, Nannan

    2012-10-01

    A previous study identified 3 nonsynonymous and 6 synonymous mutations in the entire mosquito sodium channel of Culex quinquefasciatus, the prevalence of which were strongly correlated with levels of resistance and increased dramatically following insecticide selection. However, it is unclear whether this is unique to this specific resistant population or is a common mechanism in field mosquito populations in response to insecticide pressure. The current study therefore further characterized these mutations and their combinations in other field and permethrin selected Culex mosquitoes, finding that the co-existence of all 9 mutations was indeed correlated with the high levels of permethrin resistance in mosquitoes. Comparison of mutation combinations revealed several common mutation combinations presented across different field and permethrin selected populations in response to high levels of insecticide resistance, demonstrating that the co-existence of multiple mutations is a common event in response to insecticide resistance across different Cx. quinquefasciatus mosquito populations.

  11. Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels.

    Science.gov (United States)

    Geffeney, Shana; Brodie, Edmund D; Ruben, Peter C; Brodie, Edmund D

    2002-08-23

    Populations of the garter snake Thamnophis sirtalis have evolved geographically variable resistance to tetrodotoxin (TTX) in a coevolutionary arms race with their toxic prey, newts of the genus Taricha. Here, we identify a physiological mechanism, the expression of TTX-resistant sodium channels in skeletal muscle, responsible for adaptive diversification in whole-animal resistance. Both individual and population differences in the ability of skeletal muscle fibers to function in the presence of TTX correlate closely with whole-animal measures of TTX resistance. Demonstration of individual variation in an essential physiological function responsible for the adaptive differences among populations is a step toward linking the selective consequences of coevolutionary interactions to geographic and phylogenetic patterns of diversity.

  12. Conduction abnormalities and ventricular arrhythmogenesis: The roles of sodium channels and gap junctions

    Directory of Open Access Journals (Sweden)

    Gary Tse

    2015-12-01

    Full Text Available Ventricular arrhythmias arise from disruptions in the normal orderly sequence of electrical activation and recovery of the heart. They can be categorized into disorders affecting predominantly cellular depolarization or repolarization, or those involving action potential (AP conduction. This article briefly discusses the factors causing conduction abnormalities in the form of unidirectional conduction block and reduced conduction velocity (CV. It then examines the roles that sodium channels and gap junctions play in AP conduction. Finally, it synthesizes experimental results to illustrate molecular mechanisms of how abnormalities in these proteins contribute to such conduction abnormalities and hence ventricular arrhythmogenesis, in acquired pathologies such as acute ischaemia and heart failure, as well as inherited arrhythmic syndromes.

  13. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.

    Science.gov (United States)

    Patel, Reesha R; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Cummins, Theodore R

    2016-08-01

    Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces

  14. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    Science.gov (United States)

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  15. Effect of orphanin FQ and morphine on sodium channel current in somatosensory area of rat cerebral cortex

    Institute of Scientific and Technical Information of China (English)

    Lei Yang; Yurong Li; Shuwei Jia; Yunhong Zhang; Lanwei Cui; Lihui Qu

    2007-01-01

    BACKGROUND: Some experiments have demonstrated that injecting orphanin FQ (OFQ) into lateral ventricle, which can obviously decrease the pain threshold. It is indicated that OFQ is an anti-opiate substance. However, whether OFQ has effects on sensory neuron ion channel in cerebral cortex needs to be further studied.OBJECTIVE: To investigate the effects of OFQ, morphine or their combination on sodium channel current of somatosensory neurons in rat cerebral cortex.DESIGN: Repeated measurement trial.SETTING: Department of Physiology, Harbin Medical University.MATERIALS: Fifty healthy Wistar rats, aged 12-16 days, of either gender, were provided by the Experimental Animal Center, Second Hospital Affiliated to Harbin Medical University. OFQ was purchased from Sigma-Aldrich Company, and morphine was provided by the Shenyang First Pharmaceutical Factory.PC2C patch clamp amplifier and LabmasterTLlwere purchased from Yibo Life Science Instrument Co.,Ltd.of Huazhong University of Science and Techgnology.METHODS: This experiment was carried out in the Department of Physiology (provincial laboratory),Harbin Medical University between January 2005 and May 2006. Cortical neurons were acutely isolated from rats, and prepared into cell suspension following culture. ①Sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by whole-cell Patch clamptechnique after 50 nmol/L OFQ being added to extracellular fluid.②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by the same method after 20 I mol/L morphine being added to extracellular fluid, and then the change of sodium channel current was recorded after 50 nmol/L OFQ being added.MAIN OUTCOME MEASURES: The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex following the administration of OFQ, morphine separately or their combination

  16. Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

    Science.gov (United States)

    Butterworth, Michael B; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M; Thibodeau, Patrick H

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  17. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  18. Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1

    Directory of Open Access Journals (Sweden)

    Thomas Scior

    2015-01-01

    Full Text Available The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4 was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii site-directed mutagenesis (SDM, as well as (iii electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA was expressed and tested on cells of hamster ovary (CHO. The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β proteins” (Ig-like, Ig resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another “all β protein” structure in complex with an irreversible bound protein as well as a reversible protein–protein interface (our “Rosetta Stone” effect. This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

  19. Modulatory effect of auxiliary β1 subunit on Nav1.3 voltage-gated sodium channel expressed in Xenopus oocyte

    Institute of Scientific and Technical Information of China (English)

    WANG Ying-wei; CHENG Zhi-jun; TAN Hong; XIA Yi-meng; REN Rong-rong; DING Yu-qiang

    2007-01-01

    @@ Voltage-gated sodium channels play an important role in the generation and propagation of action potentials in excitable cells. They are composed of a pore-forming α subunit and auxiliary β subunits. To date,nine subtypes of the α subunit, designated Nav 1.1 to Nav1.9, have been shown to form functional sodium channels.

  20. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  1. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  2. Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species.

    Science.gov (United States)

    Ojha, Navin K; Nematian-Ardestani, Ehsan; Neugebauer, Sophie; Borowski, Benjamin; El-Hussein, Ahmed; Hoshi, Toshinori; Leipold, Enrico; Heinemann, Stefan H

    2014-05-01

    Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

  3. Targeting voltage-gated sodium channels for treatment for chronic visceral pain

    Institute of Scientific and Technical Information of China (English)

    Fei-Hu Qi; You-Lang Zhou; Guang-Yin Xu

    2011-01-01

    Voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability, and their abnormal activity is related to several pathological processes, including cardiac arrhythmias, epilepsy, neurodegenerative diseases, spasticity and chronic pain. In particular, chronic visceral pain, the central symptom of functional gastrointestinal disorders such as irritable bowel syndrome, is a serious clinical problem that affects a high percentage of the world population. In spite of intense research efforts and after the dedicated decade of pain control and research, there are not many options to treat chronic pain conditions. However, there is a wealth of evidence emerging to give hope that a more refined approach may be achievable. By using electronic databases, available data on structural and functional properties of VGSCs in chronic pain, particularly functional gastrointestinal hypersensitivity, were reviewed. We summarize the involvement and molecular bases of action of VGSCs in the pathophysiology of several organic and functional gastrointestinal disorders. We also describe the efficacy of VGSC blockers in the treatment of these neurological diseases, and outline future developments that may extend the therapeutic use of compounds that target VGSCs. Overall, clinical and experimental data indicate that isoform-specific blockers of these channels or targeting of their modulators may provide effective and novel approaches for visceral pain therapy.

  4. Conotoxins Targeting Neuronal Voltage-Gated Sodium Channel Subtypes: Potential Analgesics?

    Directory of Open Access Journals (Sweden)

    Jeffrey R. McArthur

    2012-11-01

    Full Text Available Voltage-gated sodium channels (VGSC are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.

  5. De-novo mutations of the sodium channel gene SCN1A in alleged vaccine encephalopathy : a retrospective study

    NARCIS (Netherlands)

    Berkovic, SF; Harkin, L; McMahon, JM; Pelekanos, JT; Zuberi, SM; Wirrell, EC; Gill, DS; Iona, [No Value; Mulley, JC; Scheffer, IE

    2006-01-01

    Background Vaccination, particularly for pertussis, has been implicated as a direct cause of an encephalopathy with refractory seizures and intellectual impairment. We postulated that cases of so-called vaccine encephalopathy could have mutations in the neuronal sodium channel alpha 1 subunit gene (

  6. A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.

    Science.gov (United States)

    Kearney, J A; Plummer, N W; Smith, M R; Kapur, J; Cummins, T R; Waxman, S G; Goldin, A L; Meisler, M H

    2001-01-01

    The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

  7. Mutations in sodium channel {beta}-subunit SCN3B are associated with early-onset lone atrial fibrillation

    DEFF Research Database (Denmark)

    Olesen, Morten Salling; Jespersen, Thomas; Nielsen, Jonas Bille

    2011-01-01

    across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease...

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

    NARCIS (Netherlands)

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

    2007-01-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  10. Mutations in sodium channel {beta}-subunit SCN3B are associated with early-onset lone atrial fibrillation

    DEFF Research Database (Denmark)

    Olesen, Morten Salling; Jespersen, Thomas; Nielsen, Jonas Bille

    2011-01-01

    across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease...

  11. p.D1690N sodium voltage-gated channel α subunit 5 mutation reduced sodium current density and is associated with Brugada syndrome.

    Science.gov (United States)

    Zeng, Zhipeng; Xie, Qiang; Huang, Yuan; Zhao, Yuanyuan; Li, Weihua; Huang, Zhengrong

    2016-06-01

    Brugada syndrome (BrS) is an inherited primary arrhythmia disorder, leading to sudden cardiac death due to ventricular tachyarrhythmia, but does not exhibit clinical cardiac abnormalities. The sodium voltage-gated channel α subunit 5 (SCN5A) gene, which encodes the α subunit of the cardiac sodium channel, Nav1.5, is the most common pathogenic gene, although ≥22 BrS‑susceptibility genes have previously been identified. In the present study, a novel genetic variant (p.D1690N) localized in the S5‑S6 linker of domain IV of the Nav1.5 channels was identified in a Chinese Han family. Wild‑type (WT) and p.D1690N Nav1.5 channels were transiently over‑expressed in HEK293 cells and analyzed via the whole-cell patch clamp technique. The p.D1690N mutation significantly reduced the peak sodium current density to 23% of WT (at ‑20 mV; P<0.01), shifted steady‑state activation by 7 mV to increasingly positive potentials (P<0.01). Furthermore, prolonging of the recovery from inactivation was observed in the p.D1690N mutant. No significant change was identified in steady‑state inactivation. Thus, the mutant‑induced changes contributed to the loss of function of Nav1.5 channels, which indicates that the p.D1690N variant may have a pathogenic role in BrS.

  12. Significant natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in diabetic patients with and without nephropathy

    DEFF Research Database (Denmark)

    Andersen, Henrik; Hansen, Pernille B L; Bistrup, Claus;

    2016-01-01

    OBJECTIVE: Diabetic nephropathy is associated with aberrant glomerular filtration of serine proteases. The study was designed