Pore residues critical for μ-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis

Abstract

We have studied μ-conotoxin (μ-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amine acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to μ- CTX compared to wild-type rSkM 1 channel (IC50- = 17.5 ± 2.8 nM). E758C and D1241C were less sensitive to μ-CTX block (IC50 = 220 ± 39 nM and 112 ± 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced μ-CTX sensitivity (IC50 = 1.9 ± 0.1, 4.9 ± 0.9, and 5.5 ± 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to μ- CTX block compared to WT (p 1 μM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan- to-cysteine mutants partially or fully restored the wild-type μ-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 ± 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 ± 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for μ-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.link_to_subscribed_fulltex