Zebrafishslc4a2/ae2anion exchanger: cDNA cloning, mapping, functional characterization, and localization

Abstract

Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exonintron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h postfertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates Na ϩ -independent, electroneutral 36 Cl Ϫ /Cl Ϫ exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl Ϫ /HCO 3 Ϫ exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24 -48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function.AE2; chloride/bicarbonate exchanger CHLORIDE/BICARBONATE EXCHANGERS of the SLC4 and SLC26 gene superfamilies control intracellular pH (pH i ), cell volume, and intracellular Cl Ϫ concentration ([Cl Ϫ ]) in most body tissues (1, 37, 45). As pH regulators, they serve in basolateral HCO 3 Ϫ extrusion, contributing to epithelial acid secretion, or in apical HCO 3 Ϫ extrusion, contributing to epithelial HCO 3 Ϫ secretion. As volume regulators, they serve in basolateral Cl

High rates of HCO3– secretion and Cl– absorption against adverse gradients in the marine teleost intestine: the involvement of an electrogenic anion exchanger and H+-pump metabolon?

“…Of at least 14 different transporters capable of Na + -independent anion exchange in the gene families SLC4 (Alper 2006;Romero et al, 2004) and SLC26 (Mount and Romero, 2004), many have been identified in fish (Shmukler et al, 2005;Musch and Goldstein, 2005;Guizouarn et al, 2005;Renfro and Pritchard, 1983;Pelis and Renfro, 2003;Piermarini et al, 2002). The SLC26 gene family contains at least two (of >10) candidate genes likely to be involved in intestinal anion exchange: A3 and A6 (also known as DRA, downregulated in ademona and PAT1, putative anion transporter 1), which, in mammals, are abundant in the apical membrane of the intestinal mucosa (Mount and Romero, 2004).…”

“…Likely alternative genes from the SLC26 family include SLC26a2 and SLC26a4 (pendrin) and a possible candidate from the SLC4 family is SLC4a3 of which all are expressed apically in mammalian HCO 3 -secreting epithelia (Alper, 2006;Mount and Romero, 2004). In addition, SLC4a2, which is preferentially expressed in the basolateral region of the mammalian kidney, was recently shown to have largely apical localization in the zebrafish pronephric duct epithelium and to be stimulated by hypertonicity (Shmukler et al, 2005). Due to the apical localization of teleost SLC4a2 and the stimulation by hypertonicity, this SLC4 transporter can also be considered a likely candidate contributing to intestinal anion exchange in the marine teleost intestine.…”

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

“…Similarly, SLC26A1 has been implicated in renal sulfate transport in eel (Anguilla japonica) (Nakada et al, 2005) and rainbow trout . Little is known about the role of SLC4 Cl -/HCO 3 -exchangers in the fish kidney although SLC4A2 (AE2) has been identified in the kidney of larval zebrafish (Shmukler et al, 2005;Shmukler et al, 2008 . Second, one or more of the anion transporters may be operating in an electrogenic mode.…”

Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros

“…Expression of jagged and notch genes in the pronephros suggests that they could play an important role in patterning the pronephric nephron (Zecchin et al, 2005). Evidence for nephron patterning in the pronephros is seen in the segment-specific expression of ion transporters Shmukler et al, 2005), in the expression of receptors and transcription factors in discrete segments (Bisgrove et al, 1997;Marcos-Gutierrez et al, 1997;Van Campenhout et al, 2006), and in the disruption of segment-boundary formation in the pax2.1 (also known as pax2a -Zebrafish Information Network) mutant no isthmus (Majumdar et al, 2000). In our examination of cilia function in the kidney (Kramer-Zucker et al, 2005) we have found that two types of ciliated cells exist in the pronephros: single ciliated cells that have the morphology of typical transporting epithelial cells, and multiciliated cells that, by several criteria, appear specialized for fluid propulsion in the pronephros.…”