Molecular motions of the outer ring of charge of the sodium channel: Do they couple to slow inactivation?

Wei Xiong, Ronald A. Li, Yanli Tian, Gordon F. Tomaselli

Research output: Contribution to journalArticle

Abstract

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 β 1 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.

Original languageEnglish (US)
Pages (from-to)323-332
Number of pages10
JournalJournal of General Physiology
Volume122
Issue number3
DOIs
StatePublished - Sep 1 2003

Fingerprint

Sodium Channels
Cysteine
Potassium Channels
Xenopus
Mutagenesis
Disulfides
Oxidation-Reduction
Oocytes
Skeletal Muscle
Carbon
Brain
1,10-phenanthroline

Keywords

  • Cysteine mutagenesis
  • Disulfide bond
  • Electrophysiology
  • rNav1.4 channel

ASJC Scopus subject areas

  • Physiology

Cite this

Molecular motions of the outer ring of charge of the sodium channel : Do they couple to slow inactivation? / Xiong, Wei; Li, Ronald A.; Tian, Yanli; Tomaselli, Gordon F.

In: Journal of General Physiology, Vol. 122, No. 3, 01.09.2003, p. 323-332.

Research output: Contribution to journalArticle

Xiong, Wei ; Li, Ronald A. ; Tian, Yanli ; Tomaselli, Gordon F. / Molecular motions of the outer ring of charge of the sodium channel : Do they couple to slow inactivation?. In: Journal of General Physiology. 2003 ; Vol. 122, No. 3. pp. 323-332.
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N2 - 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 β 1 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.

AB - 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 β 1 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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KW - Electrophysiology

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