PAN, T. C.*; LIAO, B. K.; HUANG, C. J.; LIN, L. Y.; HWANG, P. P.; Univ. of North Texas, Denton; Academia Sinica, Taipei, Taiwan; Academia Sinica, Taipei, Taiwan; Academia Sinica, Taipei, Taiwan; National Taiwan Univ. and Academia Sinica, Taipei, Taiwan: Epithelial Ca2+ Channel Expression and Ca2+ Uptake in Developing Zebrafish
Ca2+ uptake mechanism has been wildly studied, but little is know of the ontogeny of epithelial calicum channels. We investigated the possible role of the ECaC (epithelial calcium channel) in the Ca2+ uptake mechanism in developing zebrafish (Danio rerio). Using rapid amplification of cDNA ends (RACE), full-length cDNA encoding the EcaC of zebrafish was cloned and sequenced. The cloned zECaC was 2578 base pairs in length, and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was expressed in all tissues examined, and began to be expressed in the skin covering the yolk sac of embryos at 24 h post-fertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac following embryonic development, and began to occur in the gill filaments at 96 hpf. Thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to the profile of ECaC expression, whole-body Ca2+ influx began to increase at 36 hpf (from 0.4±0.07 to 5.2±0.7 pmol mg-1 h-1), and Ca2+ content increased at 60 hpf (from 2.5±0.6 to 5.2±1.4 nmol mg-1) subsequently. Incubating zebrafish embryos in low-Ca2+ (0.02 mM) fresh water caused upregulation of whole-body Ca2+ influx, 299.3±49.8 pmol mg-1 h-1 compared to 177.7±42.4 pmol mg-1 h-1 in high-Ca2+ (2 mM) fresh water. zECaC expression in both gills and skin was also increased. Co-localization of zECaC mRNA and the Na+/K+-ATPase α subunit (a marker for mitochondria-rich, MR, cells) indicated that only a portion of the MR cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca2+ absorption in developing zebrafish.