Abstract

GIIIA/B μ-conotoxins block the rat skeletal muscle sodium channel (rNa_v1.4) with high affinity by binding to specific residues in the pore. However, human Na_v1.4 (hNa_v1.4) channels, which are resistant to block by GIIIA/B, have these same pore residues. We used chimera constructs, site-directed mutagenesis, and electrophysiological techniques to investigate which residues determine GIIIA/B selectivity. Exchange of serine 729 in the D2/S5-S6 linker of rat Na_v1.4 with leucine (S729L), the corresponding residue in hNa_v1.4, reduces the sensitivity of rNa_v1.4 by ∼20-fold and largely accounts for the differential sensitivity of rNa_v1.4 and hNa_v1.4 to both GIIIA and GIIIB. To determine whether D2/S5-S6 linker residues might contribute to the resistance of neuronal channels to GIIIA/B, we exchanged residues in this linker that differed between rNa_v1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa_v1.1a and rNa_v1.7, reduced the GIIIB sensitivity of rNa_v1.4 by ∼20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa_v1.4 by ∼4-fold, demonstrating that GIIIA and GIIIB have distinct interactions with the D2/S5-S6 linker. Our data indicate that naturally occurring variants in the extra-pore region of the D2/S5-S6 linker contribute to the isoform-specific sensitivity of sodium channels to GIIIA/B. Because S729 and N732 are not part of the high-affinity binding site for μ-conotoxins, these extra-pore residues probably influence the accessibility of the toxin to the binding site within the pore and/or the stability of the toxin-channel complex. Our results should aid the development of toxins that block specific neuronal sodium channel isoforms.