Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels.

摘要

To determine the roles of the individual S4 segments in domains I and II to activation and inactivation kinetics of sodium current (I_(Na)) in Navl-5, we used a tethered biotin and avidin approach after a site-directed cysteine substitution was made in the second outermost Arg in each S4 (DI-R2C and DII-R2C). We first determined the fraction of gating charge contributed by the individual S4's to maximal gating current (Q_(max)), and found that the outermost Arg residue in each S4 contributed ~19% to Q_(max) with minimal contributions by other arginines. Stabilization of the S4's in DI-R2C and DII-R2C was confirmed by measuring the expected reduction in Q_(max)- In DI-R2C, stabilization resulted in a decrease in peak I_(Na) of ~45%, while its peak current-voltage (I-V) and voltage-dependent Na channel availability (SSI) curves were nearly unchanged from wild type (WT). In contrast, stabilization of the DII-R2C enhanced activation with a negative shift in the peak I-V relationship by -7 mV and a larger -17 mV shift in the voltage-dependent SSI curve. Furthermore, its I_(Na) decay time constants and time-to-peak I_(Na) became more rapid than WT. An explanation for these results is that the depolarized conformation of DII-S4, but not DI-S4, affects the receptor for the inactivation particle formed by the interdomain linker between Dili and IV. In addition, the leftward shifts of both activation and inactivation and the decrease in G_(max) after stabilization of the DII-S4 support previous studies that showed P-scorpion toxins trap the voltage sensor of DII in an activated conformation.