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 [[sup 3] H]N-methylscopolamine in equilibrium assays is competitive and that the high affinity of UH-AH 37 for the M[sub 5] subtype, compared to M[sub 2] , 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 M[sub 2] and M[sub 5] 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 M[sub 5] receptor (M[sub 1] , M[sub 3] , and M[sub 4] 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 M[sub 2] receptor was replaced by threonine, M[sub 2] [sup 401] ala ⇒ thr, expressed enhanced affinity for pirenzepine as well as for UH-AH 37. The chick M[sub 2] 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 M[sub 2] [sup 401] ala ⇒ thr mutation.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]