EXTRAPORE RESIDUES OF THE S5-S6 LOOP OF DOMAIN-2 OF THE VOLTAGE-GATEDSKELETAL-MUSCLE SODIUM-CHANNEL (RSKM1) CONTRIBUTE TO THE MU-CONOTOXINGIIIA BINDING-SITE

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, mu-conotoxin GIIIA (mu-CTX). rSkM1 i s sensitive (IC50 = 51.4 nM) whereas hH1 is relatively resistant (IC50 = 5700 nM) to the action of the toxin, a difference in sensitivity of >100-fold. The affinity of the mu-CTX for a chimera formed from domai n 1 (D1), D2, and D3 from rSkM1 and D4 from hH1 (SSSH; S indicates ori gin of domain is skeletal muscle and H indicates origin of domain is h eart) was paradoxically increased approximately fourfold relative to t hat of rSkM1. The source of D3 is unimportant regarding the difference in the relative affinity of rSkM1 and hH1 for mu-CTX. Binding of mu-C TX to HSSS was substantially decreased (IC50 = 1145 nM). Another chime ra with a major portion of D2 deriving form hH1 showed no detectable b inding of mu-CTX (IC50 > 10 mu M). These data indicate that D1 and, es pecially, D2 play crucial roles in forming the mu-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 mu-CTX affinity with little effect on tetrodotoxin binding (less than or equal to 1.3-fold increase in affinity). Furthermore, substitu tion at Asp(730) with cysteine and exposure to Cd2+ or methanethiosulf onate reagents had no significant effect on sodium currents, consisten t with this residue not contributing to the pore.