A Novel Missense Mutation in AE1 Causing Autosomal Dominant Distal Renal Tubular Acidosis Retains Normal Transport Function but Is Mistargeted in Polarized Epithelial Cells

Abstract

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.

Primary distal renal tubular acidosis: novel findings in patients studied by next-generation sequencing

“…As the father did not have clinical or biochemical manifestations of DRTA, we concluded that it was a polymorphism. Patient 493 was heterozygous for a single described mutation G609R in the SLC4A1 gene (15). He have familial background of DRTA (Renaltube database), his affected mother who is carrier of the same mutation.…”

Regulated acid–base transport in the collecting duct

“…Mechanistically, these mutations may affect the polarized localization of AE1 at the basolateral membrane of type A intercalated cells, remain intracellular, or lose activity (6,7,39,47,93,141,150,188,189). The autosomal dominant pattern of inheritance in certain mutants is possibly due to the fact that the transporter dimerizes or that partial rerouting of mutant AE1 to the luminal membrane of type A intercalated cells may shunt normal acid secretion.…”

A New Kindred With Pseudohypoaldosteronism Type II and a Novel Mutation (564D>H) in the Acidic Motif of the WNK4 Gene

“…13 cRNA (10 ng of either NCCT or ROMK) was injected in a total volume of 100 nL per oocyte, and for coinjections involving WNK4 or one of the mutants, an additional 10 ng of WNK4 cRNA was added to the injectate. Water-injected oocytes were used as controls throughout.…”

“…11,12 Despite the interest in WNK kinases, Gordon's syndrome is extremely rare and only 4 WNK4 mutations have been reported to date in published pedigrees. 13 We describe here a new pedigree with Gordon's syndrome caused by a novel missense mutation in exon 7 on the WNK4 gene (564DϾH). The mutation lies within a motif of predominantly negatively charged amino acids that is conserved across all of the WNK genes (EPEEPEADQH), although the function of this "acidic" motif is unknown.…”