Abstract
BackgroundFreshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-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.ResultsUsing 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 Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.ConclusionThe present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.
Highlights
Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells
According to the phylogenetic analysis, these transporters were annotated as zPMCA1a, zPMCA1b, zPMCA2, zPMCA3a, zPMCA3b, zPMCA4 (Figure 3a, and in standard nomenclature ATP2B1a, ATP2B1b, ATP2B2, ATP2B3a, ATP2B3b, and ATP2B4, respectively) and zNCX1a, zNCX1b, zNCX2a, zNCX2b, zNCX3, zNCX4a, and zNCX4b (Figure 3b, in standard nomenclature SLC8a1a, SLC8a1b, SLC8a2a, SLC8a2b, SLC8a3, SLC8a4a, and SLC8a4b, respectively). zNCX1a and zNCX1b were previously described and named zNCX1h and zNCX1n, respectively [25], and zNCX2a, zNCX3, zNCX4a, and zNCX4b have been published [17,18]
According to the hydropathy analysis, putative transmembrane domains were predicted for zPMCA (Figures 1, 4a), whereas Na+/Ca2+ exchanger (NCX) has hydrophobic segments and 9 putative transmembrane domains (Figures 2, 4b) due to an intracellular helix front transmembrane domain 6 and a P loop-like structure of hydrophobic segment 8, which contained the amino acid sequence "GIG" [26]
Summary
Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. Following entry of Ca2+ through apical epithelial Ca2+ channels (ECaC, TRPV5, and/or TRPV6), Ca2+ is bound to calbindins that facilitate diffusion to the basolateral membrane, and it is extruded via the plasma membrane Ca2+-ATPase (PMCA) and/or Na+/Ca2+ exchanger (NCX). In this way, net transepithelial Ca2+ absorption from the luminal compartment (or environment) to the plasma is accomplished. Low-Ca2+ fresh water causes upregulation of the whole-body Ca2+ influx and zECaC expression in mitochondrion-rich (MR) cells of both gills and skin, providing molecular evidence for the role of ECaC in fish Ca2+ uptake [4]. No convincing evidence has been presented demonstrating the existence and involvement of PMCA and NCX in fish Ca2+ uptake mechanisms
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