Nonidentity of the α-Neurotoxin Binding Sites on the Nicotinic Acetylcholine Receptor Revealed by Modification in α-Neurotoxin and Receptor Structures

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Abstract
α-Neurotoxins constitute a large family of polypeptides that bind with high affinity to the nicotinic acetylcholine receptor (nAChR). Using a recombinant DNA-derived α-neurotoxin (Naja mossambica mossambica, NmmI) and mouse muscle nAChR expressed transiently on the surface of HEK 293 cells, we have delineated residues involved in the binding interaction on both the α-neurotoxin and the receptor interface. Several of the studied NmmI mutations, including two residues conserved throughout the α-neurotoxin family (K27 and R33), resulted in substantial decreases in the binding affinity. We have also examined 23 mutations located on the receptor α subunit and have identified 4 positions that appear to be important to NmmI recognition. These determinants represent a conserved aromatic residue (Y190), two positions where neuronal and muscle receptors differ (V188 and P197), and a negatively charged residue (D200). Unlike many of the nAChR agonists and antagonists which bind to the αδ and αγ binding sites on the receptor with different affinities, the wild-type NmmI−wild-type nAChR interaction showed a single affinity. However, by mutating critical toxin or receptor residues, we were able to produce site-selectivity between the αγ and αδ interfaces. These results suggest a nonequivalence in the binding interaction at the two sites, sensitive to discrete structural changes at key contact points on either the toxin or the receptor protein, and underscore the importance of δ and γ receptor subunits in governing binding affinity.