NONIDENTITY OF THE ALPHA-NEUROTOXIN BINDING-SITES ON THE NICOTINIC ACETYLCHOLINE-RECEPTOR REVEALED BY MODIFICATION IN ALPHA-NEUROTOXIN AND RECEPTOR STRUCTURES

alpha-Neurotoxins constitute a large family of polypeptides that bind with high affinity to the nicotinic acetylcholine receptor (nAChR). Us ing a recombinant DNA-derived alpha-neurotoxin (Naja mossambica mossam bica, NmmI) and mouse muscle nAChR expressed transiently on the surfac e of HEK 293 cells, we have delineated residues involved in the bindin g interaction on both the alpha-neurotoxin and the receptor interface. Several of the studied NmmI mutations, including two residues conserv ed throughout the alpha-neurotoxin family (K27 and R33), resulted in s ubstantial decreases in the binding affinity. We have also examined 23 mutations located on the receptor alpha subunit and have identified 4 positions that appear to be important to NmmI recognition. These dete rminants represent a conserved aromatic residue (Y190), two positions where neuronal and muscle receptors differ (V188 and P197), and a nega tively charged residue (D200). Unlike many of the nAChR agonists and a ntagonists which bind to the alpha delta and alpha gamma binding sites on the receptor with different affinities, the wild-type NmmI-wild-ty pe nAChR interaction showed a single affinity. However, by mutating cr itical toxin or receptor residues, we were able to produce site-select ivity between the alpha gamma and alpha delta interfaces. These result s suggest a nonequivalence in the binding interaction at the two sites , sensitive to discrete structural changes at key contact points on ei ther the toxin or the receptor protein, and underscore the importance of delta and gamma receptor subunits in governing binding affinity.