CHIMERIC M1/M2 MUSCARINIC RECEPTORS - CORRELATION OF LIGAND SELECTIVITY AND FUNCTIONAL COUPLING WITH STRUCTURAL MODIFICATIONS

Chimeric M1/M2 receptors were expressed in murine fibroblasts (B82) transfected with recombinant m1/m2 receptor genes. The binding affinities of a number of muscarinic antagonists and the agonist carbachol for these chimeric receptors were compared with the ligands' affinities for the M1 and M2 receptors expressed in the B82 cells. The tricyclic compounds, namely pirenzepine (PZ), 11-{[2-[(diethylamino)methyl]-1-piperidinyl]acetyl}-5,11-dihydro-6H-pyrido-[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116) and himbacine, shared a binding site between transmembrane domains VI and VII. However, the selective interaction of pirenzepine with M1 and AF-DX 116 and himbacine with M2 involved different structural regions. The high-affinity binding for 4-diphenylacetoxy-N-methylpiperidine and hexahydrosiladifenidol was confined to within loop o2 and transmembrane domains V and VI, which were clearly distinguishable from those of the tricyclic compounds. These results support the hypothesis that the ligands' stereochemical features are critical in their optimal alignment within the ligand binding pocket. The cytoplasmic i3 loop modulated the binding of carbachol such that receptors which contained the i3 domain from the M2 receptor exhibited a single high-affinity state, whereas those with the i3 domain from the M1 receptor had an additional low-affinity state for the agonist. The i3 regions are essential for the differential functional coupling of the M1 and M2 receptors to second messenger systems; however, additional upstream regions seem to be essential for a potent and efficacious activation of phospholipase C by the M1 receptor. This study provides new insight into the molecular basis of ligand selectivity.