Epithelial Ca²⁺ channel expression and Ca²⁺ uptake in developing zebrafish

The purpose of the present work was to study the possible role of the epithelial Ca²⁺ channel (ECaC) in the Ca²⁺ uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca²⁺ influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca²⁺ (0.02 mM) freshwater caused upregulation of the whole body Ca²⁺ influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na⁺-K ⁺-ATPase α-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca²⁺ absorption in developing zebrafish.