Ultra-Slow Inactivation in μ1 Na+ Channels Is Produced by a Structural Rearrangement of the Outer Vestibule

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.

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Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis

Biophysical Journal ◽

10.1016/s0006-3495(97)78768-x ◽

1997 ◽

Vol 72 (3) ◽

pp. 1204-1219 ◽

Cited By ~ 95

Author(s):

L.J. Hayward ◽

R.H. Brown ◽

S.C. Cannon

Keyword(s):

Periodic Paralysis ◽

Slow Inactivation ◽

Na Channels

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

The Journal of General Physiology ◽

10.1085/jgp.20028672 ◽

2002 ◽

Vol 120 (4) ◽

pp. 509-516 ◽

Cited By ~ 29

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.

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

Biophysical Journal ◽

10.1016/j.bpj.2010.12.2493 ◽

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

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

Biophysical Journal ◽

10.1016/j.bpj.2018.11.2105 ◽

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

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Single-channel basis of slow inactivation of Na+ channels in rat skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.3.c971 ◽

1996 ◽

Vol 271 (3) ◽

pp. C971-C981 ◽

Cited By ~ 26

Author(s):

R. L. Ruff

Keyword(s):

Skeletal Muscle ◽

Voltage Clamp ◽

Single Channel ◽

Slow Inactivation ◽

Na Channels ◽

Voltage Clamps ◽

Open Time ◽

Na Currents ◽

Fast Twitch ◽

Membrane Patch

This study examined the single-channel basis of slow inactivation of Na+ currents (INa) in rat fast-twitch skeletal muscle fibers. A loose patch voltage clamp monitored changes in the maximum inward INa as the holding potential of the membrane patch changed. On a neighboring region of extrajunctional membrane of the same fiber, a gigaohm seal patch voltage clamp recorded single-channel INa. The maximum number of simultaneously open Na+ channels among a group of current traces indicated the maximum number of excitable channels. The holding potentials of the two voltage clamps were the same. Slow inactivation did not affect the open time or conductance of single Na+ channels. The number of excitable Na+ channels reversibly decreased during development of slow inactivation of INa and increased during recovery from slow inactivation of INa. Different stimulation protocols examined whether Na+ channels had to be in the closed, open, or fast-inactivated states to enter the slow-inactivated state. Na+ channels appear to be able to enter the slow-inactivated state from the closed, open, or fast-inactivated state.

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

The Journal of General Physiology ◽

10.1085/jgp.200308881 ◽

2003 ◽

Vol 122 (3) ◽

pp. 323-332 ◽

Cited By ~ 41

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.

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