FUNCTIONAL EPITOPE ANALYSIS OF THE 2ND EXTRACELLULAR LOOP OF THE HUMAN HEART MUSCARINIC ACETYLCHOLINE-RECEPTOR

Two synthetic peptides corresponding to amino acids 172-181 and 169-19 3 of the second extracellular loop of the human M2 muscarinic receptor respectively were used to raise antibodies in rabbits. Affinity-purif ied antibodies were able not only to recognize a major band with a mol ecular weight of about 80 kDa on the electrotransferred membrane prote ins of rat ventricular membranes but also to localize the muscarinic r eceptors on the sarcolemma and t-tubules of rat, cardiomyocytes. Antib odies were also able to mimic muscarinic agonist stimulation as demons trated by a negative chronotropic effect on cultured neonatal cardiomy ocytes. In contrast with the antibodies raised against the peptide 169 -193, the antibodies against the peptide 172-181 were unable to inhibi t muscarinic ligand binding, These results suggest that the decapeptid e 172-181 contains the B-cell epitope responsible for the functional e ffect of antibodies directed against the second extracellular loop of the receptor. Coupling this peptide by cystein 177 blocks the inductio n of antibodies with pharmacological effects but induces antibodies wh ich are able to recognize the denatured receptor protein and to exert a negative chronotropic effect.