善方 文太郎

Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca ²⁺ permeability compared to that of AChRs of fast muscles. To analyse the significance of the Ca ²⁺ influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca ²⁺ -impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1–3 days post-fertilization (dpf) compared to those of zebrafish expressing Ca ²⁺ -permeable AChRs in their slow muscles. Ca ²⁺ imaging suggested that Ca ²⁺ influx via AChRs is crucial for the Ca ²⁺ response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca ²⁺ response. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca ²⁺ -permeable AChRs. These short Ca ²⁺ responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca ²⁺ influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles.

The KCNE family (KCNE1-5) is a group of single transmembrane auxiliary subunits for the voltage-gated K+ channel KCNQ1. The KCNQ1-KCNE complexes are crucial for numerous physiological processes including ventricular repolarization and K+ recycling in epithelial cells. We identified a new member of the KCNE family, "KCNE6", from zebrafish. We found that KCNE6 is expressed in the zebrafish heart and is involved in cardiac excitability. When co-expressed with KCNQ1, KCNE6 produces a slowly activating current like the slow delayed-rectifier K+ current (IKs) induced by KCNE1, despite the fact that the KCNE6 amino acid sequence has the highest similarity to that of KCNE3, which forms a constitutively open channel with KCNQ1. The kcne6 nucleotide sequences exist throughout vertebrates, including humans, although only the KCNE6 proteins of lower vertebrates, up to marsupials, can modulate KCNQ1, and it has become a pseudogene in eutherians. Our findings will facilitate a better understanding of how the KCNE family has evolved to modulate KCNQ1.

The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes (DrHCN4 and DrHCN4L; ‘Dr’ referring to Danio rerio). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in Xenopus oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in Xenopus oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs⁺, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation.

<title>Abstract</title>The mating behavior of teleost fish consists of a sequence of stereotyped actions. By observing mating of zebrafish under high-speed video, we analyzed and characterized a behavioral cascade leading to successful fertilization. When paired, a male zebrafish engages the female by oscillating his body in high frequency (<italic>quivering</italic>). In response, the female pauses swimming and bends her body (<italic>freezing</italic>). Subsequently, the male contorts his trunk to enfold the female’s trunk. This behavior is known as <italic>wrap around</italic>. Here, we found that <italic>wrap around</italic> behavior consists of two previously unidentified components. After both sexes contort their trunks, the male adjusts until his trunk compresses the female’s dorsal fin (<italic>hooking</italic>). After<italic> hooking</italic>, the male trunk slides away from the female’s dorsal fin, simultaneously sliding his pectoral fin across the female’s gravid belly, stimulating egg release (<italic>squeezing/spawning</italic>). Orchestrated coordination of <italic>spawning</italic> presumably increases fertilization success. Surgical removal of the female dorsal fin inhibited <italic>hooking</italic> and the transition to <italic>squeezing</italic>. In a neuromuscular mutant where males lack <italic>quivering</italic>, female <italic>freezing</italic> and subsequent courtship behaviors were absent. We thus identified traits of zebrafish mating behavior and clarified their roles in successful mating.