μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations 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 μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.
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