μ-Conotoxins (μ-CTXs) specifically inhibit Na+ flux by occluding the pore of voltage-gated Na+ channels. Although the three-dimensional structures of μ-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na+ channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp762 and Glu765 from domain II and Asp1241 from domain III of rat skeletal muscle Na+ channels are known to be critical for μ-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated μ-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp762 and Glu765 interact strongly with Gln14 and Arg19 but not Arg1 and that Asp1241 is tightly coupled to Lys16 but not Arg 1 or Lys11. These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na+ channels are arranged in a clockwise configuration as viewed from the extracellular surface.
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