scholarly journals The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels

1999 ◽  
Vol 113 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Vasanth Vedantham ◽  
Stephen C. Cannon
Keyword(s):  
Sodium Channel ◽  
Crucial Role ◽  
Ionic Current ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Fast Inactivation ◽  
Adult Skeletal Muscle ◽  
Α Subunit ◽  
Voltage Gated

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel α subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

Isoform-selective Effects of Isoflurane on Voltage-gated Na+ Channels

2007 ◽  
Vol 107 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Wei OuYang ◽  
Hugh C. Hemmings
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Fast Inactivation ◽  
Membrane Excitability ◽  
Voltage Gated ◽  
Hyperpolarizing Shift ◽  
Selective Effects

Abstract Background: Voltage-gated Na+ channels modulate membrane excitability in excitable tissues. Inhibition of Na+ channels has been implicated in the effects of volatile anesthetics on both nervous and peripheral excitable tissues. The authors investigated isoform-selective effects of isoflurane on the major Na+ channel isoforms expressed in excitable tissues. Methods: Rat Nav1.2, Nav1.4, or Nav1.5 α subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na+ current activation, inactivation, and recovery from inactivation were analyzed. Results: The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at −30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (−10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of −70 mV). Inhibition was greater from a holding potential of −70 mV than from −100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. Conclusions: Two principal mechanisms contribute to Na+ channel inhibition by isoflurane: enhanced inactivation due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation (Nav1.5 ≈ Nav1.4 > Nav1.2) and tonic block (Nav1.2 > Nav1.4 ≈ Nav1.5). These novel mechanistic differences observed between isoforms suggest a potential pharmacologic basis for discrimination between Na+ channel isoforms to enhance anesthetic specificity.

scholarly journals Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function

2009 ◽  
Vol 110 (3) ◽  
pp. 582-590 ◽  
Author(s):  
Wei Ouyang ◽  
Karl F. Herold ◽  
Hugh C. Hemmings
Keyword(s):  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Na Channel ◽  
Dependent Manner ◽  
Fast Inactivation ◽  
Inhaled Anesthetics ◽  
Voltage Gated ◽  
Channel Inhibition ◽  
Voltage Dependent

Background Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. Methods Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Results Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Conclusions Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

scholarly journals Slow Inactivation Does Not Affect Movement of the Fast Inactivation Gate in Voltage-gated Na+ Channels

1998 ◽  
Vol 111 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Vasanth Vedantham ◽  
Stephen C. Cannon
Keyword(s):  
Na Channel ◽  
Slow Inactivation ◽  
Na Channels ◽  
Fast Inactivation ◽  
Voltage Gated ◽  
Cytoplasmic Face ◽  
Voltage Dependent ◽  
Tightly Coupled ◽  
Exclusive Processes ◽  
A Site

Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III–IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (μl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (h∞•) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2–10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

scholarly journals Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium

2006 ◽  
Vol 127 (3) ◽  
pp. 253-265 ◽  
Author(s):  
Patrick J. Stocker ◽  
Eric S. Bennett
Keyword(s):  
Sialic Acid ◽  
Sodium Channel ◽  
Channel Gating ◽  
Sialic Acids ◽  
Na Channel ◽  
Voltage Gated Sodium Channel ◽  
Α Subunit ◽  
Rat Cardiomyocytes ◽  
Voltage Gated ◽  
Α Subunits

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an ∼10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel α subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than α subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each α subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel α subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid–dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.

P1597Two novel mutations in SCN5A gene cause enhanced inactivation and lead to Brugada syndrome

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A Zaytseva ◽  
A V Karpushev ◽  
A V Karpushev ◽  
Y Fomicheva ◽  
Y Fomicheva ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Brugada Syndrome ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Novel Mutations ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Voltage Gated ◽  
Cardiac Sodium Channel

Abstract Background Mutations in gene SCN5A, encoding cardiac potential-dependent sodium channel Nav1.5, are associated with various arrhythmogenic disorders among which the Brugada syndrome (BrS) and the Long QT syndrome (LQT) are the best characterized. BrS1 is associated with sodium channel dysfunction, which can be reflected by decreased current, impaired activation and enhanced inactivation. We found two novel mutations in our patients with BrS and explored their effect on fast and slow inactivation of cardiac sodium channel. Purpose The aim of this study was to investigate the effect of BrS (Y739D, L1582P) mutations on different inactivation processes in in vitro model. Methods Y739D and L1582P substitutions were introduced in SCN5A cDNA using site-directed mutagenesis. Sodium currents were recorded at room temperature in transfected HEK293-T cells using patch-clamp technique with holding potential −100 mV. In order to access the fast steady-state inactivation curve we used double-pulse protocol with 10 ms prepulses. To analyze voltage-dependence of slow inactivation we used two-pulse protocol with 10s prepulse, 20ms test pulse and 25ms interpulse at −100mV to allow recovery from fast inactivation. Electrophysiological measurements are presented as mean ±SEM. Results Y739D mutation affects highly conserved tyrosine 739 among voltage-gated sodium and calcium channels in the segment IIS2. Mutation L1582P located in the loop IVS4-S5, and leucine in this position is not conserved among voltage-gated channels superfamily. We have shown that Y739D leads to significant changes in both fast and slow inactivation, whereas L1582P enhanced slow inactivation only. Steady-state fast inactivation for Y739D was shifted on 8.9 mV towards more negative potentials compare with that for WT, while L1582P did not enhanced fast inactivation (V1/2 WT: −62.8±1.7 mV; Y739D: −71.7±2.3 mV; L1582P: −58.7±1.4 mV). Slow inactivation was increased for both substitutions (INa (+20mV)/INa (−100mV) WT: 0.45±0.03; Y739D: 0,34±0.09: L1582P: 0.38±0.04). Steady-state fast inactivation Conclusions Both mutations, observed in patients with Brugada syndrome, influence on the slow inactivation process. Enhanced fast inactivation was shown only for Y739D mutant. The more dramatic alterations in sodium channel biophysical characteristics are likely linked with mutated residue conservativity. Acknowledgement/Funding RSF #17-15-01292

scholarly journals μ-Conotoxin Giiia Interactions with the Voltage-Gated Na+ Channel Predict a Clockwise Arrangement of the Domains

2000 ◽  
Vol 116 (5) ◽  
pp. 679-690 ◽  
Author(s):  
Samuel C. Dudley ◽  
Nancy Chang ◽  
Jon Hall ◽  
Gregory Lipkind ◽  
Harry A. Fozzard ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Na Channel ◽  
Ion Permeation ◽  
Coupling Energy ◽  
Adult Skeletal Muscle ◽  
Voltage Gated ◽  
Single Polypeptide Chain ◽  
Anesthetic Drugs ◽  
Domain Iii ◽  
Analytical Approaches

Voltage-gated Na+ channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na+ channel (μ1) outer vestibule and selectivity filter using μ-conotoxin GIIIA (μ-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute −1.0 ± 0.1, −0.9 ± 0.3, and −1.4 ± 0.1 kcal/mol of coupling energy to the native toxin–channel complex, respectively. μ-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

Effects of Propofol on Sodium Channel-dependent Sodium Influx and Glutamate Release in Rat Cerebrocortical Synaptosomes

1997 ◽  
Vol 86 (2) ◽  
pp. 428-439 ◽  
Author(s):  
L. Ratnakumari ◽  
H. C. Hemmings
Keyword(s):  
Glutamate Release ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
General Anesthetic ◽  
Adult Rats ◽  
Sodium Influx ◽  
Voltage Dependent ◽  
Channel Dependent ◽  
Presynaptic Nerve Terminals

Background Previous electrophysiologic studies have implicated voltage-dependent Na+ channels as a molecular site of action for propofol. This study considered the effects of propofol on Na+ channel-mediated Na+ influx and neurotransmitter release in rat brain synaptosomes (isolated presynaptic nerve terminals). Methods Purified cerebrocortical synaptosomes from adult rats were used to determine the effects of propofol on Na+ influx through voltage-dependent Na+ channels (measured using 22Na+) and intracellular [Na+] (measured by ion-specific spectrofluorimetry). For comparison, the effects of propofol on synaptosomal glutamate release evoked by 4-aminopyridine (Na+ channel dependent), veratridine (Na+ channel dependent), KCi (Na+ channel independent) were studied using enzyme-coupled fluorimetry. Results Propofol inhibited veratridine-evoked 22Na+ influx (inhibitory concentration of 50% [IC50] = 46 microM; 8.9 microM free) and changes in intracellular [Na+] (IC50 = 13 microM; 6.3 microM free) in synaptosomes in a dose-dependent manner. Propofol also inhibited 4-aminopyridine-evoked (IC50 = 39 microM; 19 microM free) and veratridine (20 microM)-evoked (IC50 = 30 microM; 14 microM free), but not KCi-evoked (up to 100 microM) glutamate release from synaptosomes. Conclusions Inhibition of Na+ channel-mediated Na+ influx, increased in intracellular [Na+], and glutamate release occurred in synaptosomes at concentrations of propofol achieved clinically. These results support a role for neuronal voltage-dependent Na+ channels as a molecular target for presynaptic general anesthetic effects.

Sequence and genomic structure of the human adult skeletal muscle sodium channel α subunit gene on 17q

1992 ◽  
Vol 182 (2) ◽  
pp. 794-801 ◽  
Author(s):  
Jianzhou Wang ◽  
Cecilia V. Rojas ◽  
Jianhua Zhou ◽  
Lisa S. Schwartz ◽  
Hugh Nicholas ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Genomic Structure ◽  
Subunit Gene ◽  
Adult Skeletal Muscle ◽  
Α Subunit ◽  
Human Adult

Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II

Peptides ◽  
2015 ◽  
Vol 68 ◽  
pp. 175-182 ◽  
Author(s):  
Ying Huang ◽  
Xi Zhou ◽  
Cheng Tang ◽  
Yunxiao Zhang ◽  
Huai Tao ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Molecular Basis ◽  
Fast Inactivation ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated
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