Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor.

A number of studies have demonstrated that a major portion of the ligand binding site of the Torpedo nicotinic acetylcholine receptor is near cysteines 192 and 193 of the alpha subunit. The role of conserved tyrosine and aspartate residues within this region in ligand binding and receptor activation was investigated using a combination of site-directed mutagenesis and expression in Xenopus oocytes. Wild-type receptors are half-maximally activated (K1/2) by 20 microM acetylcholine with a Hill coefficient, n, of 1.9. Substitution of alpha Y190 and alpha Y198 with phenylalanines (alpha Y190F, alpha Y198F) or alpha D200 with asparagine (alpha D200N) shifts the K1/2 to 408, 117, and 75 microM, respectively, with no effect on the Hill coefficient. To further study the effects of these mutations on activation, the responses of the receptors to the partial agonists phenyltrimethylammonium (PTMA) and tetramethylammonium (TMA) were examined. Wild-type receptors are half-maximally activated by 73 microM PTMA and 2 mM TMA. In contrast, alpha Y190F, alpha Y198F, and alpha D200N receptors are not activated by PTMA and TMA by concentrations of up to 500 microM or 5 mM, respectively. However, PTMA and TMA do act as competitive antagonists of the mutant receptors, an indication that the binding of these compounds is not abolished by these mutations. Comparison of the the Ki values for TMA and PTMA inhibition with the K 1/2 values for TMA and PTMA activation of wild-type receptors indicates that the affinities of these compounds are similar in wild-type and mutant receptors. Therefore, alpha Y190F, alpha Y198F, and alpha D200N mutations do not significantly alter the affinity of the ligand binding site; rather, these mutations appear to interfere with the coupling of ligand binding to channel opening.