The Na channel voltage sensor associated with inactivation is localized tothe external charged residues of domain IV, S4

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 be en identified with the open-to-inactivated state kinetic transition. To inv estigate the role of the three outermost arginine amino acid residues in se gment 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 mu tations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fu sed, mammalian tsA201 cells. All four mutations had ionic currents that act ivated over the same voltage range with slope factors of their peak conduct ance-voltage (G-V) relationships similar to those of wild-type channels, al though decay of I-Na was slowest for R1C and R1Q mutant channels and fastes t for R3C mutant channels. After Na channel modification by Ap-A toxin, dec ays of I-Na were slowed to similar values for all four channel mutants. Tox in modification produced a graded effect on gating charge (Q) of mutant cha nnels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R 2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% r eduction seen for wild-type currents. Consistent with these findings, the r elationship 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 tox ins primarily inhibit gating charge associated with movement of the S4 in d omain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.