Zebrafish M(2) muscarinic acetylcholine receptor: cloning, pharmacological characterization, expression patterns and roles in embryonic bradycardia

1. A zebrafish M(2) muscarinic acetylcholine receptor (mAChR) gene was cloned. It encodes 495 amino acids in a single exon. The derived amino acid sequence is 73.5% identical to its human homologue. 2. Competitive binding studies of the zebrafish M(2) receptor and [(3)H]-NMS gave negative log dissociation constants (pK(i)) for each antagonist as follows: atropine (9.16)>himbacine (8.05)>/=4-DAMP (7.83)>AF-DX 116 (7.26)>/=pirenzepine (7.18)>/=tropicamide (6.97)>/=methoctramine (6.82)>/=p-F-HHSiD (6.67)>carbachol (5.20). The antagonist affinity profile correlated with the profile of the human M(2) receptor, except for pirenzepine. 3. Reverse transcription polymerase chain reaction and Southern blotting analysis demonstrated that the M(2) mAChR mRNA levels increased during the segmentation period (12 h post-fertilization; h.p.f.) in zebrafish. By whole-mount in situ hybridization, the M(2) mAChR was first detectable in the heart, vagus motor ganglion, and vagus sensory ganglion at 30, 48 and 60 h.p.f., respectively. 4. The muscarinic receptor that mediates carbachol (CCh)-induced bradycardia was functionally mature at 72 h.p.f. The effect of CCh-induced bradycardia was antagonized by several muscarinic receptor antagonists with the order of potency (pIC(50) values): atropine (6.76)>methoctramine (6.47)>himbacine (6.10)>4-DAMP (5.72)>AF-DX 116 (4.77), however, not by pirenzepine, p-F-HHSiD, or tropicamide (<10 micro M). 5. The effect of CCh-induced bradycardia was abolished completely before 56 h.p.f. by M(2) RNA interference, and the bradycardia effect gradually recovered after 72 h.p.f. The basal heart rate was increased in embryos injected with M(2) mAChR morpholino antisense oligonucleotide (M(2) MO) and the effect of CCh-induced bradycardia was abolished by M(2) MO in a dose-dependent manner. In conclusion, the results suggest that the M(2) mAChR inhibit basal heart rate in zebrafish embryo and the M(2) mAChR mediates the CCh-induced bradycardia.