Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells

BACKGROUND: Freshwater fish absorb Ca(2+ )predominantly from ambient water, and more than 97% of Ca(2+ )uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca(2+ )uptake in gill MR cells, Ca(2+ )passively enters the cytosol via the epithelium Ca(2+ )channel (ECaC), and then is extruded into the plasma through the basolateral Na(+)/Ca(2+ )exchanger (NCX) and plasma membrane Ca(2+)-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data. RESULTS: Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na(+)-K(+)-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca(2+ )uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca(2+)-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca(2+ )levels, while zpmcas and zncxs remained steady. CONCLUSION: The present study provides molecular evidence for the specific isoforms of Ca(2+ )transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca(2+ )uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.