Extrapore Residues of the S5-S6 Loop of Domain 2 of the Voltage-Gated Skeletal Muscle Sodium Channel (rSkM1) Contribute to the μ-Conotoxin GIIIA Binding Site

Abstract

The tetradomain voltage-gated sodium channels from rat skeletal muscle (rSkM1) and from human heart (hH1) possess different sensitivities to the 22-amino-acid peptide toxin, μ-conotoxin GIIIA ( μ-CTX). rSkM1 is sensitive (IC 50 = 51.4 nM) whereas hH1 is relatively resistant (IC 50 = 5700 nM) to the action of the toxin, a difference in sensitivity of >100-fold. The affinity of the μ-CTX for a chimera formed from domain 1 (D1), D2, and D3 from rSkM1and D4 from hH1 (SSSH; S indicates origin of domain is skeletal muscle and H indicates origin of domain is heart) was paradoxically increased approximately fourfold relative to that of rSkM1. The source of D3 is unimportant regarding the difference in the relative affinity of rSkM1 and hH1 for μ-CTX. Binding of μ-CTX to HSSS was substantially decreased (IC 50 = 1145 nM). Another chimera with a major portion of D2 deriving form hH1 showed no detectable binding of μ-CTX (IC 50 > 10 μM). These data indicate that D1 and, especially, D2 play crucial roles in forming the μ-CTX receptor. Charge-neutralizing mutations in D1 and D2 (Asp 384, Asp 762, and Glu 765) had no effect on toxin binding. However, mutations at a neutral and an anionic site (residues 728 and 730) in S5-S6/D2 of rSkM1, which are not in the putative pore region, were found to decrease significantly the μ-CTX affinity with little effect on tetrodotoxin binding (≤1.3-fold increase in affinity). Furthermore, substitution at Asp 730 with cysteine and exposure to Cd 2+ or methanethiosulfonate reagents had no significant effect on sodium currents, consistent with this residue not contributing to the pore.