Mutations in SLC4A1, encoding the chloride-bicarbonate exchanger AE1, cause distal renal tubular acidosis (dRTA), a disease of defective urinary acidification by the distal nephron. In this study we report a novel missense mutation, G609R, causing dominant dRTA in affected members of a large Caucasian pedigree who all exhibited metabolic acidosis with alkaline urine, prominent nephrocalcinosis, and progressive renal impairment. To investigate the potential disease mechanism, the consequent effects of this mutation were determined. We first assessed anion transport function of G609R by expression in Xenopus oocytes. Western blotting and immunofluorescence demonstrated that the mutant protein was expressed at the oocyte cell surface. Measuring chloride and bicarbonate fluxes revealed normal 4,4-diisothiocyanostilbene-2,2-disulfonic acidinhibitable anion exchange, suggesting that loss-offunction of kAE1 cannot explain the severe disease phenotype in this kindred. We next expressed epitopetagged wild-type or mutant kAE1 in Madin-Darby canine kidney cells. In monolayers grown to polarity, mutant kAE1 was detected subapically and at the apical membrane, as well as at the basolateral membrane, in contrast to the normal basolateral appearance of wildtype kAE1. These findings suggest that the seventh transmembrane domain that contains Gly-609 plays an important role in targeting kAE1 to the correct cell surface compartment. They confirm that dominant dRTA is associated with non-polarized trafficking of the protein, with no significant effect on anion transport function in vitro, which remains an unusual mechanism of human disease.
Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.
The endangered Asian elephant is found today primarily in protected areas. We characterized 18 dinucleotide microsatellite loci in this species. Allelic diversity ranged from three to eight per locus, and observed heterozygosity ranged from 0.200 to 0.842 in a wild population. All loci were in Hardy-Weinberg equilibrium, but linkage disequilibrium was detected between two loci in the wild, but not in the zoo elephants. These loci will be useful for the population-level studies of this species.
Anion exchanger 1 (AE1, 6 band 3, SLC4A1) is a bicarbonate transporter involved in maintaining acid-base homeostasis in the human body (1). There are two human forms of AE1, erythroid (eAE1) and kidney (kAE1). eAE1 or band 3, is the major integral membrane glycoprotein of the erythrocyte, where it serves dual roles of Cl Ϫ /HCO 3 Ϫ exchange and cytoskeletal anchorage to red cell membranes (2). kAE1 is the basolateral Cl Ϫ /HCO 3 Ϫ exchanger of the acid-secreting ␣-intercalated cell of the kidney distal tubule (3). Transcription of eAE1 in erythroid precursors is under the control of an erythroid-specific promoter upstream of exon 1, whereas renal transcription arises from a distinct promoter within intron 3 of the AE1 gene (4). Thus, the resultant kidney transcript encodes the kAE1 polypeptide lacking 65 amino acids present at the N terminus of human eAE1 (5). This structural alteration causes major functional differences between eAE1 and kAE1. The N terminus of eAE1 interacts with many proteins, including ankyrin, proteins 4.1, and glycolytic enzymes (3, 6), whereas the N terminus of kAE1 does not bind to these proteins (7,8).The three-dimensional structure of the N-terminal 43-kDa cytoplasmic domain of eAE1 revealed a globular structure, composed of 11 -strands and 10 ␣-helical segments arranged as an N-terminal interaction domain and a C-terminal dimerization domain (2), but no structure is available for kAE1. Residues 58 -68 of eAE1 form the first -strand in the cytoplasmic domain. Loss of a central strand of -sheet in kAE1 may thus greatly alter the globular structure of the cytosolic domain, thereby altering its protein interactions. Furthermore, the identity of kAE1-binding protein(s) in ␣-intercalated cells remains unknown. Previously a protein called kanadaptin (kidney anion exchanger adaptor protein) was reported to interact with mouse N-terminal kAE1 but not to eAE1 (9). Human kanadaptin does not, however, interact with human kAE1 and localizes predominantly to the nucleus (10). This leads to the question: * This work was supported in part by Canadian Institutes of Health Research operating grants (to J. R. C. and R. A. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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