Journal Article

Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel

Isabelle Deschênes, Eric Trottier and Mohamed Chahine

in Cardiovascular Research

Published on behalf of European Society of Cardiology

Volume 42, issue 2, pages 521-529
Published in print May 1999 | ISSN: 0008-6363
Published online May 1999 | e-ISSN: 1755-3245 | DOI: http://dx.doi.org/10.1016/S0008-6363(99)00064-4
Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel

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Abstract

The conserved isoleucine–phenylalanine–methionine (IFM) hydrophobic cluster located in the III–IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate in these channels. Objectives: The aim of our study was to probe the contribution of each amino acids of the IFM cluster to the inactivation. Methods: A combination of site-directed mutagenesis, cysteine covalent modification and electrophysiological recording techniques were used to elucidate the role of isoleucine1485 and methionine1487 on hH1 sodium channels expressed in tsA20l cells. Results: Mutant I1485C behaves like mutant F1486C studied earlier: producing an incomplete inactivation (residual current), a slowing and change in the voltage-dependence of the time constants of current decay, a shift of the steady-state inactivation to more depolarized voltages, and a faster recovery from inactivation than the wild-type hH1. The electrophysiological parameters of mutant M1487C are similar to those of wild-type hH1 except for the presence of a residual current. Exposure of the cytoplasmic surface of the mutants to MTS reagents MTSES, MTSET and MTSBn further disrupted inactivation. In order to explain differences in the amplitude of the sustained currents recorded in the presence of MTSES or MTSET, we studied the effects of exposure of mutants I1485C, F1486C and M1487C to acidic and basic pH in the absence and presence of MTSES and MTSET. The effects of MTSES [negatively charged (−)] and MTSET (+) on the amplitude of the residual current of mutant F1486C were modulated by changes in intracellular pH. Conclusion: Isoleucine1487 and methionine1485, which surround phenylalanine1487 contribute to stabilizing the inactivation particle for fast inactivation.

Keywords: Heart; Cellular; Electrophysiology; Molecular biology arrhythmia (mechanisms); Ion-channel; Membrane currents; Membrane transport; Na-channel

Journal Article.  5201 words.  Illustrated.

Subjects: Cardiovascular Medicine

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