Zebrafish M2 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 [H-3]-NMS gave negative log dissociation constants (pK(i)) for each antagonist as follows: atropine (9.16) > himbacine (8.05) greater than or equal to4-DAMP (7.83) > AF-DX 116 (7.26) greater than or equal to pirenzepine (7.18) greater than or equal to tropicamide (6.97) greater than or equal to methoctramine (6.82) greater than or equal to 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 muM). 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.