scholarly journals Slow Inactivation Does Not Block the Aqueous Accessibility to the Outer Pore of Voltage-gated Na Channels

2002 ◽  
Vol 120 (4) ◽  
pp. 509-516 ◽  
Author(s):  
Arie F. Struyk ◽  
Stephen C. Cannon
Keyword(s):  
Reaction Rates ◽  
Slow Inactivation ◽  
Na Channels ◽  
Fast Inactivation ◽  
Voltage Gated ◽  
State Dependent ◽  
Gating Mechanism ◽  
Dependent Change ◽  
Outer Pore ◽  
Inactivation Gating

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.

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.

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 State-dependent Block of Wild-type and Inactivation-deficient Na+ Channels by Flecainide

2003 ◽  
Vol 122 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ging Kuo Wang ◽  
Corinna Russell ◽  
Sho-Ya Wang
Keyword(s):  
Time Constant ◽  
Open Channel ◽  
Inhibitory Concentration ◽  
Antiarrhythmic Agent ◽  
Na Channels ◽  
Fast Inactivation ◽  
Wild Type ◽  
Open Channel Block ◽  
State Dependent ◽  
Na Currents

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM−1s−1 and 12.2 s−1, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.

scholarly journals Enhancement of Slow Inactivation of Voltage-Gated Na+ Channels by Ranolazine

2011 ◽  
Vol 100 (3) ◽  
pp. 421a
Author(s):  
Ryoko Hirakawa ◽  
Lynda V. Liu ◽  
John C. Shryock ◽  
Luiz Belardinelli ◽  
Sridharan Rajamani
Keyword(s):  
Slow Inactivation ◽  
Na Channels ◽  
Voltage Gated

HMJ-53A accelerates slow inactivation gating of voltage-gated K+ channels in mouse neuroblastoma N2A cells

2008 ◽  
Vol 54 (7) ◽  
pp. 1128-1135 ◽  
Author(s):  
Chia-Chia Chao ◽  
Jeffrey Shieh ◽  
Sheng-Chu Kuo ◽  
Bor-Tsang Wu ◽  
Mann-Jen Hour ◽  
...  
Keyword(s):  
Slow Inactivation ◽  
K Channels ◽  
Voltage Gated ◽  
Mouse Neuroblastoma ◽  
N2a Cells ◽  
Inactivation Gating

scholarly journals In Search of a Molecular Mechanism for Slow Inactivation in Voltage-Gated Na Channels using the SCAM Technique in D2-S6 of hNav1.4

2019 ◽  
Vol 116 (3) ◽  
pp. 389a
Author(s):  
John P. O'Reilly ◽  
Kevin Bokum ◽  
Jonathan Beard ◽  
Penny Shockett
Keyword(s):  
Molecular Mechanism ◽  
Slow Inactivation ◽  
Na Channels ◽  
Voltage Gated

scholarly journals A Structural Rearrangement in the Sodium Channel Pore Linked to Slow Inactivation and Use Dependence

2000 ◽  
Vol 116 (5) ◽  
pp. 653-662 ◽  
Author(s):  
Boon-Hooi Ong ◽  
Gordon F. Tomaselli ◽  
Jeffrey R. Balser
Keyword(s):  
Conformational Changes ◽  
Structural Rearrangement ◽  
Side Chain ◽  
Na Channel ◽  
Slow Inactivation ◽  
Na Channels ◽  
Loss Of Function ◽  
Conformational States ◽  
Hek 293 ◽  
Outer Pore

Voltage-gated sodium (Na+) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na+ channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na+ channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (IM; τ ∼ 30 ms) and slower (IS; τ ∼ 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the IM and IS kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na+ channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

scholarly journals Molecular Motions of the Outer Ring of Charge of the Sodium Channel

2003 ◽  
Vol 122 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Wei Xiong ◽  
Ronald A. Li ◽  
Yanli Tian ◽  
Gordon F. Tomaselli
Keyword(s):  
Structural Rearrangement ◽  
Outer Ring ◽  
Na Channel ◽  
Slow Inactivation ◽  
Redox Catalyst ◽  
Cysteine Mutagenesis ◽  
Charged Ring ◽  
Outer Pore ◽  
Inactivation Gating

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain β1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the α carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.

scholarly journals Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4–S5 linker in activation and slow inactivation

2021 ◽  
Vol 118 (28) ◽  
pp. e2102285118
Author(s):  
Jinglei Xiao ◽  
Vasyl Bondarenko ◽  
Yali Wang ◽  
Antonio Suma ◽  
Marta Wells ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Specific Binding ◽  
Drug Binding ◽  
Dependent Manner ◽  
Slow Inactivation ◽  
Voltage Gated Sodium Channel ◽  
Structural Investigations ◽  
Voltage Gated ◽  
Cell Functions ◽  
Gating Mechanism

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4–S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4–S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4–S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed “door-wedge” action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

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