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). Using a recombinant DNA-derived alpha-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 alpha-neurotoxin and the receptor interface. Several of the studied NmmI mutations, including two residues conserved throughout the alpha-neurotoxin family (K27 and R33), resulted in substantial 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 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 alphadelta and alphagamma 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 alphagamma and alphadelta 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 delta and gamma receptor subunits in governing binding affinity.
Ackermann
E J
EJ
Department of Pharmacology 0636, University of California, San Diego, La Jolla, California 92093, USA.
Taylor
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D007700:Kinetics; D051379:Mice; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D002038:Bungarotoxins; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins