The Na Channel Voltage Sensor Associated with Inactivation Is Localized to the External Charged Residues of Domain IV, S4

Abstract

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.

Pharmacology of the Na v 1.1 domain IV voltage sensor reveals coupling between inactivation gating processes

“…6A). The first step initiates fast inactivation and corresponds to the transition of VSDIV from the resting state to the activated state, a step that accounts for the majority of gating charge movement (15,(32)(33)(34)(35). The second, more weakly voltage-dependent, transition moves the activated VSDIV to an immobilized state that is coupled to selectivity filter collapse and slow inactivation, as has been proposed for other voltage-gated channels (14,17,36).…”

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

“…A conservative substitution of Phe 15 by Trp had no effect, whereas substitution to Ala decreased the potency of Lqh2 for rNa v 1.2a by 5.5-fold ( Fig. 1 and supplemental Table S1 and Fig.…”

“…The channel site of interaction with scorpion ␣-toxins, named neurotoxin receptor site-3 (12), is shared also by structurally unrelated toxins from sea anemone and spider venoms (13,14), which raises questions as to its architecture and boundaries. Based on the findings that site-3 toxins eliminate a gating charge component associated with the movement of D4/S4 (15,16), and that this segment plays a critical role in coupling channel inactivation to activation (17), scorpion ␣-toxins were postulated to inhibit channel inactivation by hindering the outward movement of this segment during depolarization (9).…”

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

“…Identification of the amino acid residues in the IVS3-S4 loop that are required for high affinity binding of ␣-scorpion toxins led to the proposal that the voltage sensor in domain IV is primarily involved in fast inactivation (13). Consistent with that model, mutations of the gating charges in the IVS4 segment completely occlude the effects of ␣-scorpion toxins on gating current (27). Fluorescent labeling studies show that the four voltage sensors in the different domains are functionally specialized.…”