PHARMACOLOGICAL CHARACTERIZATION OF HUMAN M1 MUSCARINIC ACETYLCHOLINE-RECEPTORS WITH DOUBLE MUTATIONS AT THE JUNCTION OF TM-VI AND THE 3RD EXTRACELLULAR DOMAIN

A mutant human m5 receptor containing the mutations of Ser465 to Tyr a nd Thr466 to Pro showed constitutive activity. By replacing the equiva lent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and dis played enhanced responses to classical muscarinic agonists with signif icantly increased potencies. Choline, a normal component of growth med ia, showed an efficacy comparable to acetylcholine and carbachol at Hm 1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicoti nic agonist, exhibited partial agonist activity at human mi wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) recep tors. I-Hyoscyamine inhibited the activities of choline and methylcarb achol. Muscarinic antagonists displayed small reductions in binding af finities, although muscarinic agonists showed greatly increased bindin g affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, in cluding choline and methylcarbachol, showed multiple affinity states a t Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The hi gh affinity binding sites far acetylcholine, arecoline and choline wer e shifted in the presence of GppNHp. These results suggest that Hm1(Se r388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.