USE OF CHIMERIC MUSCARINIC RECEPTORS TO INVESTIGATE EPITOPES INVOLVEDIN ALLOSTERIC INTERACTIONS

All five (m1-m5) muscarinic receptors are sensitive to allosteric regu lation, but gallamine is considerably more potent in slowing the disso ciation of N-[H-3]methylscopolamine (NMS) from the m2 subtype than fro m the m3 or m5 subtypes. To study the structural basis for the prefere nce of gallamine for the m2 subtype, we evaluated [H-3]NMS-gallamine i nteractions with chimeric receptors in which segments of the m5 recept or were systematically replaced with the corresponding m2 sequence. Su bstitutions that included the sixth transmembrane domain and third ext racellular loop resulted in marked increases in the potency of gallami ne, but substitutions that did not include these regions were without effect. A similar substitution was investigated using m2/m3 chimeric r eceptors, in which a segment extending from the middle of the sixth tr ansmembrane domain to the carboxyl terminus was exchanged. As with the m2/m5 constructs, substitution of the m2 carboxyl-terminal segment in to the m3 subtype significantly increased the potency of gallamine. Fu rthermore, the converse substitution reduced the potency of gallamine dramatically, to approximately that seen for the m3 subtype itself. It appears that this portion of the receptor is a critical determinant f or the binding of gallamine and/or the allosteric interactions between gallamine and [H-3]NMS.