Orientation of μ-Conotoxin PIIIA in a Sodium Channel Vestibule, Based on Voltage Dependence of Its Binding

Mutant cycle analysis has been used in previous studies to constrain possible docking orientations for various toxins. As an independent test of the bound orientation of mu-conotoxin PIIIA, a selectively targeted sodium channel pore blocker, we determined the contributions to binding voltage dependence of specific residues on the surface of the toxin. A change in the apparent valence (z delta) of the block, which is associated with a change of a specific toxin charge, reflects a change in the charge movement within the transmembrane electric field as the toxin binds. Toxin derivatives with charge-conserving mutations (R12K, R14K, and K17R) showed z delta values similar to those of wild type (0.61 +/- 0.01, mean +/- S.E.M.). Charge-changing mutations produced a range of responses. Neutralizing substitutions for Arg14 and Lys17 showed the largest reductions in z delta values, to 0.18 +/- 0.06 and 0.20 +/- 0.06, respectively, whereas unit charge-changing substitutions for Arg12, Ser13, and Arg20 gave intermediate values (0.24 +/- 0.07, 0.33 +/- 0.04, and 0.32 +/- 0.05), which suggests that each of these residues contributes to the dependence of binding on the transmembrane voltage. Two mutations, R2A and G6K, yielded no significant change in z delta. These observations suggest that the toxin binds with Arg2 and Gly6 facing the extracellular solution, and Arg14 and Lys17 positioned most deeply in the pore. In this study, we used molecular dynamics to simulate toxin docking and performed Poisson-Boltzmann calculations to estimate the changes in local electrostatic potential when individual charges were substituted on the toxin's surface. Consideration of two limiting possibilities suggests that most of the charge movement associated with toxin binding reflects sodium redistribution within the narrow part of the pore.