Novel structural determinants of mu-conotoxin (GIIIB) block in rat skeletal muscle (mu 1) Na+ channels

mu-Conotoxin (mu-CTX) specifically occludes the pore of voltage-dependent N a+ channels. In the rat skeletal muscle Na+ channel (mu 1), we examined the contribution of charged residues between the P loops and S6 in all four do mains to mu-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for mu-CTX blo ck by similar to 100-fold (wild-type = 22.3 +/- 7.0 nM; D762C = 2558 +/- 25 0 nM; E765C = 2020 +/- 379 nM). Restoration or reversal of charge by extern al modification of the cysteine-substituted channels with methanethiosulfon ate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosu lfonate ethylammonium (MTSEA)) did not affect mu-CTX block (D762C: IC50, MT SEA+ = 2165.1 +/- 250 nM; IC50, MTSES- = 2753.5 +/- 456.9 nM; E765C: IC50, MTSEA+ = 2200.1 +/- 550.3 nM; IC50, (MTSES-) = 3248.1 +/- 2011.9 nM) compar ed with their unmodified counterparts. In contrast, the charge-conserving m utations D762E (IC50 = 21.9 +/- 4.3 nM) and E765D (IC50 = 22.0 +/- 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 +/- 687.9 nM) and E765K (IC50 = 4202.7 +/- 1088.0 nM) destabilized CL-CTX block even further, suggesting a prominent electrostati c component of the interactions between these DII residues and mu-CTX. Kine tic analysis of mu-CTX block. reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with litt le changes in association (k(on)) rates. We conclude that the acidic residu es at positions 762 and 765 are key determinants of mu-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MT SEA side chain to modify mu-CTX sensitivity places steric constraints on th e sites of toxin interaction.