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Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization

¹Molecular and Vascular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center, ³Hematology Division, Brigham and Women's Hospital, ⁴Hematology Division and ⁷Howard Hughes Medical Institute, Children's Hospital, ⁵Renal Unit, Massachusetts General Hospital, and Departments of ²Medicine and ⁶Pediatrics, Harvard Medical School, Boston, Massachusetts

Submitted 28 March 2005 ; accepted in final form 16 May 2005

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 exon-intron 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⁺-independent, electroneutral ³⁶Cl^–/Cl^– 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^–/HCO₃^– 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.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Zebrafish Ae2 mediates Cl–/Cl– exchange. A: 36Cl– uptake into oocytes previously injected with water or with 3 or 10 ng cRNA encoding Ae2 or mAE2a. B: time course of 36Cl– efflux from representative oocytes previously injected with water () or with cRNA encoding Ae2 () or mAE2a (). Bath pH was 7.4 in the presence (ND-96) and absence of Cl–. C: 36Cl– efflux rate constants of oocytes expressing Ae2 or mAE2a measured sequentially in ND-96, Cl–-free medium, and finally in ND-96 containing 200 µM DIDS. D: DIDS concentration dependence of 36Cl– efflux rate constants in oocytes expressing Ae2 compared with mAE2a. Values in all bar graphs are means ± SE for (n) oocytes. Oocytes in B–D were injected with 2.7 ng ae2 cRNA or 3 ng of mAE2a cRNA. 36Cl– efflux rate constants in bar graphs reflect subtraction of values measured in water-injected oocytes.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Zebrafish Ae2 mediates Cl–/HCO3– exchange. A: changes in pHi measured in representative BCECF-loaded Xenopus laevis oocytes in the presence of CO2/HCO3– during bath Cl– removal and restoration. Oocytes were previously injected with water or with 3 or 10 ng ae2 cRNA as indicated. B: mean rates of Cl–/HCO3– exchange ± SD presented as proton-equivalent flux (JH+).

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