Aromatics at the murine nicotinic receptor agonist binding site:: mutational analysis of the αY93 and αW149 residues

1. Two aromatic residues of the muscle nicotinic receptor putative agonist binding site, a tyrosine in position alpha 93 and a tryptophan in position alpha 149, were mutated to phenylalanine and the effects of the mutations on receptor properties were investigated using single-channel patch clamp. 2. The alpha Y93F mutation reduced the receptor affinity by similar to4-fold and the channel opening rate constant by 48-fold. The alpha W149F mutation reduced the receptor affinity by similar to 12-fold and the channel opening rate constant by 93-fold. 3. The kinetic properties of hybrid receptors that contained one wild-type and one mutated a subunit were also examined. Only one type of hybrid receptor activity was detected. The hybrid receptors had a channel opening rate constant intermediate to those of the wild-type and mutant receptors. It was concluded that the ligand binding sites in the mutated muscle nicotinic receptor contributed equally to channel gating. In the case of the alpha W149F mutation, the presence of the mutation in one of the binding sites had no effect on the binding properties of the other, non-mutated, site. 4. The mutant channel opening and closing rate constants were also estimated in the presence of tetramethylammonium. The data suggested significant interaction between the acetyl group of acetylcholine and the alpha Y93 residue.