Abstract

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 μM to 10 mM), a novel K⁺ current with both delayed rectifying and inward rectifying properties (I_KTMA) was activated in single canine atrial myocytes. 4-Aminopyridine (0.05–2 mM) also induced a K⁺ current (I_K4AP) with characteristics similar to but distinct from those of I_KTMA. Both I_KTMAand I_K4AP were abolished by 1 μM atropine. I_K4AP, but not I_KTMA, was minimized by treatment with pertussis toxin. I_KTMA 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 I_K4AP, but the antagonists for other mAChR subtypes had no effects on I_K4AP. By comparison, I_KACh (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.