Abstract

The five muscarinic receptors (m1-m5), although structurally closely related, can be distinguished pharmacologically by the use of subtype-selective ligands. Various tricyclic muscarinic antagonists, including the AF-DX derivative AQ-RA 741 and the alkaloid himbacine, for example, have been shown to display up to 200-fold higher affinities for m2 and m4 than for m5 receptors. On the other hand, antagonists such as sila-hexocyclium and the pirenzepine derivative UH-AH 37 exhibit lower affinities for m2 than for m5 and all other muscarinic receptors. To identify receptor epitopes that contribute to the subtype selectivities of these antagonists, we prepared a series of chimeric m2/m5 muscarinic receptors in which regions of the m5 receptor were systematically replaced with the homologous regions of the m2 receptor. AQ-RA 741, himbacine, and sila-hexocyclium bound to the various chimeric receptors, expressed in COS-7 cells, with affinity profiles indicative of multiple receptor domains contributing to the subtype selectivities of these antagonists. On the other hand, the higher affinity of UH-AH 37 for m5 than for m2 receptors appears to be largely dependent on a short stretch of 31 amino acids comprising most of transmembrane region VI and the third extracellular loop, a region that does not contribute to the subtype selectivity of AQ-RA 741 and himbacine. Our data indicate that different receptor epitopes are involved in conferring subtype selectivity on structurally different muscarinic antagonists.