PORE RESIDUES CRITICAL FOR MU-CTX BINDING TO RAT SKELETAL-MUSCLE NA+ CHANNELS REVEALED BY CYSTEINE MUTAGENESIS

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sod ium channel (rSkM1) currents in which single amino acids within the po re (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sens itivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to CL-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptopha n 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). D 400C and Y401C also showed statistically significant yet modest (appro ximately twofold) changes in sensitivity to mu-CTX block compared to W T (p < 0.05). Application of the negatively charged, sulfhydryl-reacti ve compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the t oxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the ot her hand, the positively charged methanethiosulfonate-ethylammonium (M TSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 m u M) 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 try ptophan's indole group is a determinant of toxin binding. In support o f this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was si milar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results dem onstrate that charge at positions 758 and 1241 are important for mu-CT X toxin binding and further suggest that the tryptophan residues withi n the pore in domains I, III, and IV negatively influence toxin-channe l interaction.