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, mu-conotoxin GIIIA (mu-CTX). rSkM1 is 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 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 mu-CTX. Binding of mu-CTX to HSSS was substantially decreased (IC50 = 1145 nM). Another chimera with a major portion of D2 deriving form hH1 showed no detectable binding of mu-CTX (IC50 > 10 microM). These data indicate that D1 and, especially, D2 play crucial roles in forming the mu-CTX receptor. Charge-neutralizing mutations in D1 and D2 (Asp384, Asp762, and Glu765) 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 (</=1.3-fold increase in affinity). Furthermore, substitution at Asp730 with cysteine and exposure to Cd2+ or methanethiosulfonate reagents had no significant effect on sodium currents, consistent with this residue not contributing to the pore.

Critical Molecular Determinants of Voltage-Gated Sodium Channel Sensitivity to μ-Conotoxins GIIIA/B

“…Chimera inserts were confirmed by sequencing the junctions. The chimera channel showed toxin sensitivity (data not shown) identical to that of hNa v 1.4, in general agreement with published results (Chahine et al, 1998a) that domain 2 determines GIIIA/B sensitivity. The sequence encoding hNa v 1.4 domain 4 was isolated as a SacII/EcoRI fragment and used to replace the remaining rNa v 1.4 domain 4 in the chimera.…”

“…Therefore, we compared the sequences of rNa v 1.4 and hNa v 1.4 in this region to identify residues that might contribute to the differential sensitivity of the two channels to -conotoxins. The residues that Chahine et al (1998b) mutated in rNa v 1.4 (A728 and D730) are conserved in hNa v 1.4; however, the D2/S5-S6 linker in these two channels differs by two amino acid residues (Fig. 2).…”

ConusVenoms: A Rich Source of Novel Ion Channel-Targeted Peptides

“…It is known that -GIIIA interacts with the pore region of the ion channel and therefore competes with TTX for binding to site I. Structure-function studies have revealed a rather complex interaction of -GIIIA and -GIIIB with the ion channel pore, with several amino acids at different positions of the peptide contributing to the high affinity of binding. These data indicate that binding sites for TTX and -conotoxins overlap, but are not identical (42,129); some mutations that have strong effects on the TTX sensitivity of Na v 1.4 lead to only a minor increase in the IC 50 of -GIIIA to the channel (19,21,175).…”

The Selectivity Filter of the Voltage-gated Sodium Channel Is Involved in Channel Activation

“…In theory, a part of -CTX R13Q protruding into the outer pore could act as a splint in the vestibule, preventing a pore collapse as the molecular event underlying ultra-slow inactivation. Alternatively, the toxin might, by virtue of multiple interactions with the outer surface of the channel (56,57), act as a molecular scaffold, thereby stabilizing the structure of the outer vestibule.…”