PREDOMINANT INTERACTIONS BETWEEN MU-CONOTOXIN ARG-13 AND THE SKELETAL-MUSCLE NA+ CHANNEL LOCALIZED BY MUTANT CYCLE ANALYSIS

High-affinity mu-conotoxin block of skeletal muscle Na+ channels depen ds on an arginine at position 13 (Arg-13). To understand both the mech anism of toxin interaction and the general structure of its binding si te in the channel mouth, we examined by thermodynamic mutant cycle ana lysis the interaction between the critical Arg-13 and amino acid resid ues known to be in the channel's outer vestibule. Arg-13 interacts spe cifically with domain II Glu-758 with energy of about -3.0 kcal/mol, i ncluding both electrostatic and nonelectrostatic components, and with Glu-403 with energy of about -2.0 kcal/mol. Interactions with the othe r charged residues in the outer vestibule were shown to be almost enti rely electrostatic, because these interactions were maintained when Ar g-13 was replaced by lysine. These results place the bound Arg-13 at t he channel mouth adjacent to the P (pore) loops of domains I and II. D istance estimates based on interaction energies suggest that the charg ed vestibule residues are in relative positions similar to those of th e Lipkind-Fozzard vestibule model [Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13]. Kinetic analysis suggests that Arg-13 in teractions are partially formed in the ligand-channel transition state .