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

Abstract

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 [ 3H]-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 μM). 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.