Critical molecular determinants of voltage-gated sodium channel sensitivity to μ-conotoxins GIIIA/B

GIIIA/B mu-conotoxins block the rat skeletal muscle sodium channel (rNa(v) 1.4) with high affinity by binding to specific residues in the pore. However, human Na(v)1.4 (hNa(v)1.4) 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(v)1.4 with leucine (S729L), the corresponding residue in hNa(v)1.4, reduces the sensitivity of rNa(v)1.4 by similar to20-fold and largely accounts for the differential sensitivity of rNa(v)1.4 and hNa(v)1.4 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(v)1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa(v)1.1a and rNa(v)1.7, reduced the GIIIB sensitivity of rNa(v)1.4 by similar to20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa(v)1.4 by similar to4-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 mu-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.