Site-directed mutagenesis implicates a threonine residue in TM6 in the subtype selectivities of UH-AH 37 and pirenzepine at muscarinic receptors

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.