A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence

B. H. Ong, Gordon F. Tomaselli, J. R. Balser

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

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 (I(M);τ ~ 30 ms) and slower (I(S);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 I(M) and I(s) 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.

Original languageEnglish (US)
Pages (from-to)653-661
Number of pages9
JournalJournal of General Physiology
Volume116
Issue number5
DOIs
StatePublished - Nov 20 2000
Externally publishedYes

Fingerprint

Sodium Channels
Cysteine
Lidocaine
Voltage-Gated Sodium Channels
HEK293 Cells
Patch-Clamp Techniques
Muscle Cells
Skeletal Muscle
Mutation
methanethiosulfonate ethylammonium

Keywords

  • Cysteine mutagenesis
  • Electrophysiology
  • Gating
  • Lidocaine
  • Local anesthetic

ASJC Scopus subject areas

  • Physiology

Cite this

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence. / Ong, B. H.; Tomaselli, Gordon F.; Balser, J. R.

In: Journal of General Physiology, Vol. 116, No. 5, 20.11.2000, p. 653-661.

Research output: Contribution to journalArticle

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N2 - 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 (I(M);τ ~ 30 ms) and slower (I(S);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 I(M) and I(s) 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.

AB - 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 (I(M);τ ~ 30 ms) and slower (I(S);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 I(M) and I(s) 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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