Identification and characterization of multiple subtypes of muscarinic acetylcholine receptors and their physiological functions in canine hearts.

M2 receptors have long been believed to be the only functional subtype of muscarinic acetylcholine receptor (mAChR) in the heart, although recent studies have provided evidence for the presence of other subtypes. We performed a detailed study to clarify this issue. In the presence of tetramethylammonium (1 microM to 10 mM), a novel K+ current with both delayed rectifying and inward rectifying properties (IKTMA) was activated in single canine atrial myocytes. 4-Aminopyridine (0.05-2 mM) also induced a K+ current (IK4AP) with characteristics similar to but distinct from those of IKTMA. Both IKTMA and IK4AP were abolished by 1 microM atropine. IK4AP, but not IKTMA, was minimized by treatment with pertussis toxin. IKTMA was markedly decreased by 4-diphenylacetoxy-N-methylpiperidine methiodide (a selective antagonist for M3 subtype) but was not altered by pirenzepine (for M1), methoctramine (for M2), and tropicamide (for M4). Tropicamide substantially reduced IK4AP, but the antagonists for other mAChR subtypes had no effects on IK4AP. By comparison, IKACh (ACh-induced K+ current) was significantly depressed by methoctramine but was unaltered by other antagonists. Results from displacement binding of [methyl-3H]N-scopolamine methyl chloride with pirenzepine, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methiodide, or tropicamide revealed the coexistence of multiple mAChR subtypes in canine atrium. Cloning of cDNA fragments and detection of mRNAs coding for M2, M3, and M4 provided further supporting evidence. Our results suggest that 1) multiple subtypes of mAChRs (M2/M3/M4) coexist in the dog heart and 2) different subtypes of mAChRs are coupled to different K+ channels. Our findings represent the first functional evidence for the physiological role of cardiac M3 and M4 receptors.