The structural basis for the selectivity of the antagonist UH-AH 37 at human muscarinic acetylcholine receptors was investigated by expressing mutant receptors in COS-7 cells. Previous studies have demonstrated that the interaction between UH-AH 37 and [3H]N-methylscopolamine in equilibrium assays is competitive and that the high affinity of UH-AH 37 for the M5 subtype, compared to M2, is due to an epitope in the sixth transmembrane domain (TM6) or the third outer loop of the receptor. By mutating each nonconserved residue in this region of M2 and M5 to its counterpart in the other receptor, we identified a threonine residue in the middle of TM6 uniquely responsible for the higher affinity of the M5 receptor (M1, M3, and M4 receptors also carry a threonine at that location and also have high affinity for UH-AH 37). The mutant receptor in which the corresponding alanine of the M2 receptor was replaced by threonine, M2401 ala → thr, expressed enhanced affinity for pirenzepine as well as for UH-AH 37. The chick M2 receptor, which expresses anomalously high affinity for pirenzepine, differs from its mammalian counterparts by the presence of a threonine at this position. Affinities of AF-DX 116 and 4-DAMP, as well as the allosteric potency of UH-AH 37, were not sensitive to the M2401 ala → thr mutation. Copyright (C) 2000 S. Karger AG, Basel.
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