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

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 acid-inhibitable anion exchange, suggesting that loss-of-function of kAE1 cannot explain the severe disease phenotype in this kindred. We next expressed epitope-tagged 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 wild-type 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. 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 acid-inhibitable anion exchange, suggesting that loss-of-function of kAE1 cannot explain the severe disease phenotype in this kindred. We next expressed epitope-tagged 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 wild-type 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. Distal renal tubular acidosis (dRTA) 1The abbreviations used are: dRTA, distal renal tubular acidosis; ddRTA, autosomal dominant dRTA; HEK, human embryonic kidney; MDCK, Madin-Darby canine kidney; HA, hemagglutinin; PBS, phosphate-buffered saline. is a disease of defective urinary acidification characterized by impaired H+ secretion into the urine leading to metabolic acidosis. This disorder is usually accompanied by hypokalemia, metabolic bone disease, nephrocalcinosis, and/or nephrolithiasis (1Rodriguez-Soriano J. Pediatr. Nephrol. 2000; 14: 1121-1136Google Scholar, 2Batlle D. Ghanekar H. Jain S. Mitra A. Annu. Rev. Med. 2001; 52: 471-484Google Scholar, 3Karet F.E. J. Am. Soc. Nephrol. 2002; 13: 2178-2184Google Scholar). Mutations in SLC4A1 (MIM 109270), encoding the polytopic chloride-bicarbonate exchanger known as AE1, have been reported as the sole genetic cause of autosomal dominant dRTA (ddRTA) (MIM #179800) (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar, 5Karet F.E. Gainza F.J. Gyory A.Z. Unwin R.J. Wrong O. Tanner M.J. Nayir A. Alpay H. Santos F. Hulton S.A. Bakkaloglu A. Ozen S. Cunningham M.J. di Pietro A. Walker W.G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6337-6342Google Scholar, 6Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Google Scholar, 7Weber S. Soergel M. Jeck N. Konrad M. Pediatr. Nephrol. 2000; 15: 201-204Google Scholar, 8Sritippayawan S. Kirdpon S. Vasuvattakul S. Wasanawatana S. Susaengrat W. Waiyawuth W. Nimmannit S. Malasit P. Yenchitsomanus P.T. Pediatr. Nephrol. 2003; 18: 644-648Google Scholar, 9Cheidde L. Vieira T.C. Lima P.R. Saad S.T. Heilberg I.P. Pediatrics. 2003; 112: 1361-1367Google Scholar) and also rarely cause autosomal recessive dRTA (10Tanphaichitr V.S. Sumboonnanonda A. Ideguchi H. Shayakul C. Brugnara C. Takao M. Veerakul G. Alper S.L. J. Clin. Investig. 1998; 102: 2173-2179Google Scholar, 11Vasuvattakul S. Yenchitsomanus P.T. Vachuanichsanong P. Thuwajit P. Kaitwatcharachai C. Laosombat V. Malasit P. Wilairat P. Nimmannit S. Kidney Int. 1999; 56: 1674-1682Google Scholar, 12Ribeiro M.L. Alloisio N. Almeida H. Gomes C. Texier P. Lemos C. Mimoso G. Morle L. Bey-Cabet F. Rudigoz R.C. Delaunay J. Tamagnini G. Blood. 2000; 96: 1602-1604Google Scholar, 13Bruce L.J. Wrong O. Toye A.M. Young M.T. Ogle G. Ismail Z. Sinha A.K. McMaster P. Hwaihwanje I. Nash G.B. Hart S. Lavu E. Palmer R. Othman A. Unwin R.J. Tanner M.J. Biochem. J. 2000; 350: 41-51Google Scholar, 14Yenchitsomanus P.T. Vasuvattakul S. Kirdpon S. Wasanawatana S. Susaengrat W. Sreethiphayawan S. Chuawatana D. Mingkum S. Sawasdee N. Thuwajit P. Wilairat P. Malasit P. Nimmannit S. Am. J. Kidney Dis. 2002; 40: 21-29Google Scholar). Although several different SLC4A1 mutations have been found to be involved in this disease, the molecular mechanisms of pathogenesis for these mutations are not completely defined. In humans, the two main sites of AE1 expression are in erythrocyte cell membranes and, in the kidney, at the basolateral surface of α-intercalated cells in the collecting duct segment of the nephron. The encoded proteins are both products of SLC4A1, but because different promoters initiate transcription, they differ at their N termini, the renal isoform (kAE1) missing the first 65 amino acids found in the erythrocyte protein. Reported SLC4A1 mutations associated with ddRTA include missense alterations of Arg-589 (R589H, R589S, and R589C) (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar, 5Karet F.E. Gainza F.J. Gyory A.Z. Unwin R.J. Wrong O. Tanner M.J. Nayir A. Alpay H. Santos F. Hulton S.A. Bakkaloglu A. Ozen S. Cunningham M.J. di Pietro A. Walker W.G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6337-6342Google Scholar, 6Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Google Scholar, 7Weber S. Soergel M. Jeck N. Konrad M. Pediatr. Nephrol. 2000; 15: 201-204Google Scholar, 8Sritippayawan S. Kirdpon S. Vasuvattakul S. Wasanawatana S. Susaengrat W. Waiyawuth W. Nimmannit S. Malasit P. Yenchitsomanus P.T. Pediatr. Nephrol. 2003; 18: 644-648Google Scholar) and S613F (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar); A>L889X (9Cheidde L. Vieira T.C. Lima P.R. Saad S.T. Heilberg I.P. Pediatrics. 2003; 112: 1361-1367Google Scholar); and a complex mutation resulting in an 11-amino acid truncation at the C terminus, R901X (5Karet F.E. Gainza F.J. Gyory A.Z. Unwin R.J. Wrong O. Tanner M.J. Nayir A. Alpay H. Santos F. Hulton S.A. Bakkaloglu A. Ozen S. Cunningham M.J. di Pietro A. Walker W.G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6337-6342Google Scholar). These mutations have been studied for their anion transport function and protein trafficking by expression in Xenopus oocytes and cultured mammalian cells, for example human embryonic kidney (HEK) and Madin-Darby canine kidney (MDCK) cells. When mutant R589H, R589C, or S613F AE1 proteins were expressed in Xenopus oocytes, they reached the cell surface and exhibited at worst only mildly reduced anion exchange activity (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar, 6Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Google Scholar). Impaired trafficking of mutant R589H, R589C, and R589S kAE1 proteins was demonstrated when expressed in HEK 293 cells, and co-expression of wild-type kAE1 with these mutant proteins also revealed a dominant negative effect (15Quilty J.A. Li J. Reithmeier R.A. Am. J. Physiol. Renal Physiol. 2002; 282: F810-F820Google Scholar). Furthermore, it has been suggested that the R901X mutant retained normal anion exchange activity in Xenopus oocytes but failed to express at the cell surface of unpolarized MDCK cells (16Toye A.M. Bruce L.J. Unwin R.J. Wrong O. Tanner M.J. Blood. 2002; 99: 342-347Google Scholar) and HEK 293 cells (17Quilty J.A. Cordat E. Reithmeier R.A. Biochem. J. 2002; 368: 895-903Google Scholar). From all of these data, it has been concluded that mutant AE1 proteins maintain sufficient anion transport function but are defective in targeting to the cell surface. These results suggest that the explanation for AE1 mutations and ddRTA is not simple loss-of-function. As kAE1 is specifically located in the basolateral membrane of α-intercalated cells in the distal nephron, an alternative reasonable hypothesis is that kAE1 mutations do not cause complete intracellular retention but instead result in mistargeting to the apical rather than basolateral membrane of these cells (13Bruce L.J. Wrong O. Toye A.M. Young M.T. Ogle G. Ismail Z. Sinha A.K. McMaster P. Hwaihwanje I. Nash G.B. Hart S. Lavu E. Palmer R. Othman A. Unwin R.J. Tanner M.J. Biochem. J. 2000; 350: 41-51Google Scholar, 18Tanner M.J. Nature. 1996; 382: 209-210Google Scholar). However, almost all the protein-targeting studies that demonstrated impaired trafficking of mutant kAE1 were conducted in non-polarized cells, so it is possible that these proteins may behave differently in α-intercalated cells. In support of this, we have recently demonstrated that kAE1-R901X is aberrantly targeted to the apical surface of polarized MDCK cells, which emphasizes the importance of polarized cell models for targeting studies of the membrane proteins (19Devonald M.A. Smith A.N. Poon J.P. Ihrke G. Karet F.E. Nat. Genet. 2003; 33: 125-127Google Scholar). In this study, we identified a novel missense mutation of the AE1 gene in a Caucasian family with dRTA. This mutation was further examined for its transport function in Xenopus oocytes and its trafficking in polarized MDCK cells. Subjects—Several members of a Caucasian kindred with autosomal dominant dRTA were studied (Fig. 1A). All affected individuals were determined to have metabolic acidosis with alkaline urine, nephrocalcinosis, and progressive renal impairment. Control subjects consisted of 30 unrelated, unaffected individuals from the same ethnic background. Mutation Screening of SLC4A1—Each coding exon of the kidney isoform of SLC4A1 (kAE1), including its flanking exon-intron boundaries, was individually amplified from genomic DNA of study patients. These PCR products were sequenced using ABI Prism® BigDye™ terminator cycle sequencing (Applied Biosystems) on an ABI377 instrument. Identified sequence alterations were confirmed in family members and assessed in 30 controls by allele-specific PCR (20Newton C.R. Graham A. Heptinstall L.E. Powell S.J. Summers C. Kalsheker N. Smith J.C. Markham A.F. Nucleic Acids Res. 1989; 17: 2503-2516Google Scholar, 21Kwok S. Kellogg D.E. McKinney N. Spasic D. Goda L. Levenson C. Sninsky J.J. Nucleic Acids Res. 1990; 18: 999-1005Google Scholar) using primers BD3x15R-ASA-G (5′-GTC GGG TCA TCG AGG ACT ACG-3′) or BD3x15R-ASA-A (5′-GTC GGG TCA TCG AGG ACT ACA-3′) and BD3x15F (5′-CAG GGA TGG GGT AGA GGT AGT C-3′). Plasmid Construction for Expression in Xenopus Oocytes—A cDNA clone containing full-length kAE1 constructed in pHM6 was used as template for PCR amplification using the Expand™ long template PCR system (Roche Applied Science) and specific primers to introduce a HindIII site, a Kozak consensus sequence, start codon, and hemagglutinin (HA) tag upstream of the second codon of kAE1, and a HindIII site downstream of the stop codon of the gene. This 2.6-kb PCR product was digested with HindIII and cloned into a vector containing the 5′- and 3′-untranslated regions of Xenopus β-globin (gift of Cecilia Canessa, Yale University) for expression in Xenopus oocytes. This wild-type kAE1 clone, pXG-kAE1, was used as a template for site-directed mutagenesis (QuikChange™ site-directed mutagenesis kit, Stratagene) to make a mutant clone, pXG-kAE1G609R. The sequences of these two cDNA constructs were verified by direct and were with of using with the to the and in Xenopus kAE1 and Xenopus oocytes were with of or an of and were in at for was to the of and A.M. J. Biol. Chem. 1989; Scholar). of oocytes were in containing at for of oocytes were with in at for of the the of the with oocytes were in a oocyte was to an containing of of was in a of the to which the oocytes were were also for so that the chloride be from a of the specific Although bicarbonate from oocytes not be the from of oocytes was assessed to confirm the function of kAE1. oocytes were in a for at This was and with of were at and were with and the of the was using a The is by the is the A.K. A. R.A. R.J. J. Physiol. 1999; Scholar). and Western oocyte membranes were to I. F. M.T. Am. J. Physiol. 1989; Scholar, S.J. P. M. J.P. J. Biol. Chem. 1996; Scholar, D.L. L. J. Biol. 2002; Scholar). of or oocytes were using in containing complete (Roche were at for at to all and were at for 30 to a membrane were in of and protein was determined by protein were at for to on a and to membranes and were in with and with a at in containing for in membranes were for with in specific proteins were detected using the to the with or mutant kAE1 Xenopus oocytes were for in at The was with in at for were in and on were on a and on These were with PBS, at for and with at was in in PBS, were with at in the for further in PBS, were using was on a Plasmid Construction for Expression in MDCK and were used as template for amplification using long PCR and which HindIII and sites to the 5′- and of the kAE1 These PCR products were digested and into vector to and The of these complete constructs was confirmed by DNA Plasmid DNA for was with the and cells were cultured in with and and at with cells in were with of or using (Roche To cells were with and were cultured in this for of cells were and at on MDCK cells were with for to for of kAE1 protein by immunofluorescence as cells on membranes were with and in containing and at for with PBS, were in and in at for by further in was with in for and were with of (Roche Applied for with was by with for These were were with of to and with of to for 30 in the with and with were as with the expression of protein was examined by a (Fig. at the of with and was found to have a metabolic acidosis with alkaline urine and prominent with In with and of and two of a of renal tubular acidosis was with was and with and progressive renal from In an from unaffected were to revealed disease with renal and renal has well a of this kindred that several first have also been with (Fig. 1A). Renal is a prominent in this as is renal impairment. is a renal at of kAE1 G609R sequence studies of SLC4A1 in the revealed a to in exon GGG to AGG to a novel missense G609R mutation (Fig. and mutations were found in allele-specific amplification was to this sequence in members of the DNA from was amplified in two with a of or which the same PCR product of of or The results that the mutation with disease phenotype in family members and (Fig. The mutation was in all affected individuals but both from an unaffected family and from 30 Expression of kAE1 in Xenopus expression of the G609R kAE1 wild-type or mutant encoding full-length human kAE1 an tag were first expressed in Xenopus oocytes. Western blotting of membrane from oocytes using an demonstrated a protein of molecular to kAE1, in both wild-type and mutant oocytes, which was in oocytes (Fig. was also to confirm the expression of wild-type and mutant kAE1 at the membrane of the Xenopus The of kAE1 in oocytes with wild-type or mutant kAE1 expression of the protein at the cell surface in both (Fig. this immunofluorescence was in oocytes with or in negative controls was These results that the mutant G609R protein normal and was expressed at the cell surface of Xenopus oocytes in to wild-type of kAE1 in the membrane of oocytes wild-type kAE1 and oocytes with or were and detected but was in of kAE1 in Xenopus investigate the mutation kAE1 transport chloride and bicarbonate were of by or oocytes with or of a specific of AE1 J. and of is in These results significant wild-type or mutant with but no significant wild-type and mutant of by wild-type and mutant kAE1 proteins was and of We also determined by of oocytes with wild-type or mutant which were with for the The by both wild-type and mutant kAE1 proteins were by but were not different from of was of and with of in a of oocytes with kAE1 as a exchanger D. L. Annu. Rev. Biochem. Scholar) the basolateral membrane of α-intercalated cells, we to confirm this function of the mutant protein by Although not be from oocytes, we a to its from of oocytes. As this revealed results to the The by oocytes with wild-type or mutant kAE1 was and of these that this kAE1 mutant protein normal anion transport of kAE1 in MDCK cells with or kAE1 demonstrated different expression of these proteins (Fig. As reported (19Devonald M.A. Smith A.N. Poon J.P. Ihrke G. Karet F.E. Nat. Genet. 2003; 33: 125-127Google wild-type kAE1 was at the basolateral surface of cells grown to on (Fig. In the G609R mutant protein was in a that the basolateral surface and with apical with the surface (Fig. in this study were the genetic cause of ddRTA in a large family who exhibited and renal and in the of a novel SLC4A1 This at is all known AE1 gene family an of importance of this Gly-609 is to the seventh transmembrane domain of the polytopic AE1 protein, its J. Biol. Chem. 2003; Scholar). Gly-609 is also located to two missense mutations have been in Arg-589 and the importance of this of the of the The mutation reported results in of a amino acid by a is not effect this have on the of this but studies that it not of the protein. that this is the its by and of or targeting but this a for at In the potential disease mechanism, of this G609R kAE1 mutation on anion transport were assessed by a of Xenopus oocyte and polarized cell The expression used for and trafficking studies of membrane proteins are Xenopus oocytes an expression for transport function of both because their large and and because they do not express kAE1, of the activity of the mutant proteins A.M. J. Biol. Chem. 1989; Scholar, Tanner M.J. J. Biol. Chem. Scholar). However, oocytes cannot be used for of potential polarized S.L. Annu. Rev. Physiol. 2002; Scholar). In the of a α-intercalated cell cultured mammalian cells as MDCK cells, a canine collecting duct cell that polarized have for In with it remains to these cells with kAE1 and they are not an cell for α-intercalated cells as they are from cells, the cell in the collecting studies to the of apical expression of mutant kAE1 be a with sufficient expression be but we have to been to significant expression in to this In of mutation in this disease, R589H, R589C, and R589S were reported in and (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar, 5Karet F.E. Gainza F.J. Gyory A.Z. Unwin R.J. Wrong O. Tanner M.J. Nayir A. Alpay H. Santos F. Hulton S.A. Bakkaloglu A. Ozen S. Cunningham M.J. di Pietro A. Walker W.G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6337-6342Google Scholar, 6Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Google Scholar, 7Weber S. Soergel M. Jeck N. Konrad M. Pediatr. Nephrol. 2000; 15: 201-204Google Scholar, 8Sritippayawan S. Kirdpon S. Vasuvattakul S. Wasanawatana S. Susaengrat W. Waiyawuth W. Nimmannit S. Malasit P. Yenchitsomanus P.T. Pediatr. Nephrol. 2003; 18: 644-648Google S613F was in kindred (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar). The mechanisms of these reported mutations studies concluded that anion exchange is at worst only mildly (4Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Investig. 1997; 100: 1693-1707Google Scholar, 6Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Google Scholar). has been suggested that intracellular of and R901X expressed in non-polarized cells is the dominant negative effect the of urine acidification and dominant of dRTA (15Quilty J.A. Li J. Reithmeier R.A. Am. J. Physiol. Renal Physiol. 2002; 282: F810-F820Google Scholar, J.A. Cordat E. Reithmeier R.A. Biochem. J. 2002; 368: 895-903Google Scholar). However, kAE1-R901X expression in MDCK cells grown to results in surface expression with mutant protein at the apical surface (19Devonald M.A. Smith A.N. Poon J.P. Ihrke G. Karet F.E. Nat. Genet. 2003; 33: 125-127Google Scholar) in with the findings reported on the Arg-589 have to be assessed in polarized cells, and results for the different mutations may be different because of In of AE1 are not associated with a renal suggesting that of of the encoded protein is not sufficient to explain dRTA P. Rubin H.L. J.T. L.M. L. V. J. Blood. 1996; Scholar). is possible that the mechanisms associated with different mutations may not be the but alternative for these have to The of in distal is from the of in to and members of this kindred exhibited a of and renal Although the of reported is to it is that they this with the kindred the R901X mutation mistargeting of the mutant protein is also a is to that this is because these mutations result in bicarbonate secretion into the urine rather than but this has not been to this be to urine that in affected members of this kindred and the R901X it not be reduced to the usually in dRTA. is that severe and consequent the of of trafficking of this novel G609R mutation associated with ddRTA that this in the seventh transmembrane plays an important role in correct protein a effect on anion transport As AE1 is a D. A. J.T. Blood. 2000; 96: we that both and of wild-type and mutant protein in the apical compartment. This effect of protein of kAE1, with the mutation, to impaired urine acidification by the α-intercalated cells resulting in severe disease. of mutant in of I. 2001; Scholar) is the known example of a mechanism of disease that remains with We and for