The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae

Abstract

SUMMARYAfter demonstrating phylogenetic relatedness to orthologous mammalian genes, tools were developed to investigate the roles of three members (A3, A4 and A6c) of the SLC26 anion exchange gene family in Cl–uptake and HCO3 excretion in embryos and larvae of zebrafish(Danio rerio). Whole-mount in situ hybridization revealed the presence of SLC26 mRNA in gill primordia, mesonephros and heart(slc26a3 and a4 only) at 5–9 days postfertilization(d.p.f.). SLC26A3 protein was highly expressed in lateral line neuromasts and within the gill, was localized to a sub-population of epithelial cells, which often (but not always) coexpressed Na+/K+-ATPase. SLC26 mRNA levels increased with developmental age, peaking at 5–10 d.p.f.;the largest increases in rates of Cl– uptake(\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document}) preceded the mRNA spike, occurring at 2–5 d.p.f. Raising zebrafish in water with a low [Cl–] caused marked increases in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} at 3–10 d.p.f. and was associated with increased levels of SLC26 mRNA. Raising fish in water of high [Cl–] was without effect on \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} or SLC26 transcript abundance. Selective gene knockdown using morpholino antisense oligonucleotides demonstrated a significant role for SLC26A3 in Cl– uptake in larval fish raised in control water and roles for A3, A4 and A6c in fish raised in water with low [Cl–]. Prolonged (7 days) or acute (24 h) exposure of fish to elevated (2 or 5 mmol l–1) ambient [HCO3–] caused marked increases in Cl– uptake when determined in water of normal [HCO3–] that were accompanied by elevated levels of SLC26 mRNA. The increases in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(J_{\mathrm{in}}^{\mathrm{Cl}^{-}}\) \end{document} associated with high ambient [HCO3–] were not observed in the SLC26 morphants (significant only at 5 mmol l–1HCO3– for A4 and 2 mmol l–1HCO3– for A6c). Net base excretion was markedly inhibited in the slc26a3 and a6c morphants thereby implicating these genes in Cl–/HCO3– exchange. The results suggest that under normal conditions, Cl– uptake in zebrafish larvae is mediated by SLC26A3 Cl–/HCO3– exchangers but under conditions necessitating higher rates of high affinity Cl–uptake, SlC26A4 and SLC26A6c may assume a greater role.