The outermost lysine in the S4 of domain III contributes little to the gating charge in sodium channels

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, Na(V)1.5) to the total gating charge (Q(max)). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the alpha1 subunit in fused tsA201 cells. We used the relative reduction in Q(max) caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin's ability to inhibit gating charge in mutant channels showed that R2 contributed 19-20% to the Q(max), R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.