Polymodal, High Affinity Actions of μ-Conotoxins on a Bacterial Voltage-Gated Sodium Channel

Abstract

μ-Conotoxins (μCTXs) are potent blockers of eukaryotic sodium channels, and individual members of the μCTX family are highly specific for particular Nav1 isoforms. We have begun to explore μCTX interactions with NaChBac, a prokaryotic Nav channel closely related to NavAb, whose crystal structure was recently determined (Payandeh et al, 2011, Nature 475:353). Our data reveal actions on both ion conduction and gating, consistent with polymodal actions on the pore domain.Under voltage clamp, whole-cell currents from NaChBac expressed in tsA-201 cells were reduced, up to nearly complete block, by concentrations in the pM to μM range, for wildtype toxins and derivatives of μCTX PIIIA and KIIIA. For wildtype PIIIA, dose-response data (0.001-10,000nM) yielded the following: IC50=0.005nM; maximal fraction of current blocked = 0.95; Hill coefficient = 0.7. Chen & Chung (2012, Biophys. J. 102:483) predicted an IC50 of 0.1nM for PIIIA when the NavAb channel is occupied by 2 sodium ions.Even at very low [PIIIA] (1pM), the unblocked currents showed increasing rates of inactivation as the peptides were washed in, suggesting a gating modulation, that was not tightly associated with pore block. For μCTX PIIIA (0.1nM), or KIIIA (30nM), inactivation accelerated by ∼10-fold. Substitution of key basic residues (PIIIA-R14A and KIIIA-K7A) reduced blocking potency and decreased the speeding of inactivation to less than 2-fold.Given the remarkably high selectivity and affinity that μCTXs show for certain eukaryotic Nav channels with highly asymmetric pores, it may seem surprising that they bind to symmetric bacterial Nav channels with such high affinity. However, in both cases, there is the potential for multiple electrostatic interactions to sum and yield potent binding. Our data suggest that pore-block, and acceleration of inactivation, result from distinct molecular actions of the toxin.