title: Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis.
abstract: We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino 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 mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-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 mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) 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 mu-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 mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: R A
authors.initials: RA
authors.lastname: Li
authors.forename: R G
authors.initials: RG
authors.lastname: Tsushima
authors.forename: R G
authors.initials: RG
authors.lastname: Kallen
authors.forename: P H
authors.initials: PH
authors.lastname: Backx
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D002104:Cadmium / Q000378:metabolism; D003553:Cystine / Q000737:chemistry / Q000235:genetics; D005260:Female; D066298:In Vitro Techniques; D008564:Membrane Potentials; D008978:Mollusk Venoms / Q000378:metabolism* / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism*; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: D008978:Mollusk Venoms; D009842:Oligopeptides; D011994:Recombinant Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D002104:Cadmium; D003553:Cystine