Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization.
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 (36)Cl(-)/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(-)/HCO(3)(-) 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.
Shmukler
Boris E
BE
Molecular Medicine and Renal Units, Beth Israel Deaconess Med. Ctr. E/RW763, 330 Brookline Ave., Boston, MA 02215, USA.
Kurschat
Christine E
CE
Ackermann
Gabriele E
GE
Jiang
Lianwei
L
Zhou
Yi
Y
Barut
Bruce
B
Stuart-Tilley
Alan K
AK
Zhao
Jinhua
J
Zon
Leonard I
LI
Drummond
Iain A
IA
Vandorpe
David H
DH
Paw
Barry H
BH
Alper
Seth L
SL
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins / Q000235:genetics*; D017920:Antiporters / Q000235:genetics*; D001639:Bicarbonates / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004625:Embryo, Nonmammalian; D005091:Exons / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064606:SLC4A Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D014982:Xenopus laevis; D015027:Zebrafish
D027321:Anion Transport Proteins; D017920:Antiporters; D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D018076:DNA, Complementary; D012333:RNA, Messenger; D064606:SLC4A Proteins; C579496:SLC4A2 protein, human; C579495:Slc4a2 protein, mouse