Functional epitope analysis of the second extracellular loop of the human heart muscarinic acetylcholine receptor.

Two synthetic peptides corresponding to amino acids 172-181 and 169-193 of the second extracellular loop of the human M2 muscarinic receptor respectively were used to raise antibodies in rabbits. Affinity-purified antibodies were able not only to recognize a major band with a molecular weight of about 80 kDa on the electrotransferred membrane proteins of rat ventricular membranes but also to localize the muscarinic receptors on the sarcolemma and t-tubules of rat cardiomyocytes. Antibodies were also able to mimic muscarinic agonist stimulation as demonstrated by a negative chronotropic effect on cultured neonatal cardiomyocytes. In contrast with the antibodies raised against the peptide 169-193, the antibodies against the peptide 172-181 were unable to inhibit muscarinic ligand binding. These results suggest that the decapeptide 172-181 contains the B-cell epitope responsible for the functional effect of antibodies directed against the second extracellular loop of the receptor. Coupling this peptide by cystein 177 blocks the induction of antibodies with pharmacological effects but induces antibodies which are able to recognize the denatured receptor protein and to exert a negative chronotropic effect.