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

Abstract

Slow inactivation in voltage-gated Na channels (NaChs) probably involves a complex molecular mechanism that is currently not well understood. We studied NaCh slow inactivation using the substituted cysteine accessibility method (SCAM) in the S6 segment of domain 2 (D2-S6) in the human skeletal muscle voltage-gated Na channel (hNav1.4). We produced mutants with a single cysteine substitution in D2-S6 from V788 (near the glycine gating hinge) to L797 (near the bundle crossing). Voltage-clamp recordings of Na current from these mutants were used to determine (1) the effect, if any, of the cysteine substitution on the fast kinetics (activation and steady-state fast inactivation), (2) the effect, if any, on the kinetics of slow inactivation (development, steady-state, and recovery from), and (3) the accessibility of the substituted cysteine to modification with methanethiosulfonate ethylammonium (MTSEA) when the channels were (i) at rest, (ii) fast inactivated, and (iii) slow inactivated. Most of the mutants have been produced, and several have been analyzed (others are in progress). We currently have preliminary results from three mutants - L792C, V793C, and V794C. None of these mutants demonstrated significant changes in fast gating. However, in L792, there was a significant hyperpolarization of steady-state slow inactivation, in V793C there was a significant enhancement of slow inactivation development, and in V794, there was enhancement of slow inactivation and accessibility to MTSEA modification (recorded as a non-reversible reduction in whole-cell current) during slow inactivation, which was not seen in the closed or fast inactivated states. Experiments in progress will produce more information on these and other mutants.