Mechanism of μ-Conotoxin PIIIA Binding to the Voltage-Gated Na+ Channel NaV1.4

Several subtypes of voltage-gated Na+ (Na-V) channels are important targets for pain management. mu-Conotoxins isolated from venoms of cone snails are potent and specific blockers of different Na-V channel isoforms. The inhibitory effect of mu-conotoxins on Na-V channels has been examined extensively, but the mechanism of toxin specificity has not been understood in detail. Here the known structure of m-conotoxin PIIIA and a model of the skeletal muscle channel Na(V)1.4 are used to elucidate elements that contribute to the structural basis of m-conotoxin binding and specificity. The model of Na(V)1.4 is constructed based on the crystal structure of the bacterial Na-V channel, Na(V)Ab. Six different binding modes, in which the side chain of each of the basic residues carried by the toxin protrudes into the selectivity filter of Na(V)1.4, are examined in atomic detail using molecular dynamics simulations with explicit solvent. The dissociation constants (K-d) computed for two selected binding modes in which Lys9 or Arg14 from the toxin protrudes into the filter of the channel are within 2 fold; both values in close proximity to those determined from dose response data for the block of Na-V currents. To explore the mechanism of PIIIA specificity, a double mutant of Na(V)1.4 mimicking Na-V channels resistant to m-conotoxins and tetrodotoxin is constructed and the binding of PIIIA to this mutant channel examined. The double mutation causes the affinity of PIIIA to reduce by two orders of magnitude.