NEGATIVELY CHARGED AMINO-ACID-RESIDUES IN THE NICOTINIC RECEPTOR DELTA-SUBUNIT THAT CONTRIBUTE TO THE BINDING OF ACETYLCHOLINE

In nicotinic receptors, the binding sites for acetylcholine are likely to contain negatively charged amino acid side chains that interact wi th the positively charged quaternary ammonium group of acetylcholine a nd of other potent agonists. We previously found that a 61-residue seg ment of the delta subunit contains aspartate or glutamate residues wit hin 1 nm of cysteines in the acetylcholine binding site on the alpha s ubunit. We have now mutated, one at a time, the 12 aspartates and glut amates in this segment of the mouse muscle delta subunit and have expr essed the mutant receptors in Xenopus oocytes. Both the concentration of acetylcholine eliciting half-maximal current (K(app)) and the K(i) for the inhibition by acetylcholine of alpha-bungarotoxin binding were increased 100-fold by the mutation of deltaAsp180 to Asn and 10-fold by the mutation of deltaGlu189 to Gln. These two residues, and their h omologs in the gamma and epsilon subunits, are likely to contribute to the acetylcholine binding sites.