Slc26a11, a chloride transporter, localizes with the vacuolar H + -ATPase of A-intercalated cells of the kidney
2011; Elsevier BV; Volume: 80; Issue: 9 Linguagem: Inglês
10.1038/ki.2011.196
ISSN1523-1755
AutoresJie Xu, Sharon Barone, Hong Li, Shannon Holiday, Kamyar Zahedi, Manoocher Soleimani,
Tópico(s)Connexins and lens biology
ResumoChloride has an important role in regulating vacuolar H+-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H+-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H+-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (∼72kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H+-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H+-ATPase. Thus, Slc26a11 is a partner of vacuolar H+-ATPase facilitating acid secretion in the collecting duct. Chloride has an important role in regulating vacuolar H+-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H+-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H+-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (∼72kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H+-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H+-ATPase. Thus, Slc26a11 is a partner of vacuolar H+-ATPase facilitating acid secretion in the collecting duct. SLC26(human)/Slc26(mouse) isoforms belong to a conserved family of anion transporters and are expressed in various tissues and organs, with some paralogs displaying specific tissue, cell, or subcellular expression pattern.1.Bissig M. Hagenbuch B. Stieger B. et al.Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.J Biol Chem. 1994; 269: 3017-3021PubMed Google Scholar, 2.Hastbacka J. de la Chapelle A. Mahtani M.M. et al.The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 3.Hoglund P. Haila S. Socha J. et al.Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhea.Nat Genet. 1996; 14: 316-319Crossref PubMed Scopus (322) Google Scholar, 4.Everett L.A. Glaser B. Beck J.C. et al.Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).Nat Genet. 1997; 17: 411-422Crossref PubMed Scopus (948) Google Scholar, 5.Zheng J. Shen W. He D.Z. et al.Prestin is the motor protein of cochlear outer hair cells.Nature. 2000; 405: 149-155Crossref PubMed Scopus (917) Google Scholar, 6.Lohi H. Kujala M. Kerkela E. et al.Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger.Genomics. 2000; 70: 102-112Crossref PubMed Scopus (189) Google Scholar, 7.Lohi H. Kujala M. Makela S. et al.Functional characterization of three novel tissue-specific anion exchangers SLC26A7, -A8, and -A9.J Biol Chem. 2002; 277: 14246-14254Crossref PubMed Scopus (182) Google Scholar, 8.Vincourt J.B. Jullien D. Amalric F. et al.Molecular and functional characterization of SLC26A11, a sodium-independent sulfate transporter from high endothelial venules.FASEB J. 2003; 17: 890-892Crossref PubMed Scopus (76) Google Scholar SLC26 isoforms can transport various anions, including chloride, sulfate, bicarbonate, and oxalate, with variable specificity.9.Romero M.F. Chang M.H. Plata C. et al.Physiology of electrogenic SLC26 paralogues.Novartis Found Symp. 2006; 273: 126-138Crossref PubMed Google Scholar,10.Soleimani M. Xu J. SLC26 chloride/base exchangers in the kidney in health and disease.Semin Nephrol. 2006; 26: 375-385Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Several SLC26A members function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1), SLC26A7, and SLC26A9.11.Melvin J.E. Park K. Richardson L. et al.Mouse down-regulated in adenoma (DRA) is an intestinal Cl(-)/HCO(3)(-) exchanger and is upregulated in colon of mice lacking the NHE-3 Na(+)/H(+) exchanger.J Bio Chem. 1999; 274: 22855-22861Crossref PubMed Scopus (236) Google Scholar, 12.Soleimani M. Greeley T. Petrovic S. et al.Pendrin: an apical Cl-/OH-/HCO3- exchanger in the kidney cortex.Am J Physiol Renal Physiol. 2001; 280: F356-F364Crossref PubMed Google Scholar, 13.Petrovic S. Ju X. 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Nakamura S. et al.Deletion of the chloride transporter slc26a7 causes distal renal tubular acidosis and impairs gastric acid secretion.J Biol Chem. 2009; 284: 29470-29479Crossref PubMed Scopus (68) Google Scholar, 18.Xu J. Song P. Miller M.L. et al.Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach.Proc Natl Acad Sci USA. 2008; 105: 17955-17960Crossref PubMed Scopus (77) Google Scholar SLC26A7 and SLC26A9 can also function as chloride channels.18.Xu J. Song P. Miller M.L. et al.Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach.Proc Natl Acad Sci USA. 2008; 105: 17955-17960Crossref PubMed Scopus (77) Google Scholar, 19.Chang M.H. Plata C. Zandi-Nejad K. et al.Slc26a9—anion exchanger, channel and Na+ transporter.J Membr Biol. 2009; 228: 125-140Crossref PubMed Scopus (58) Google Scholar, 20.Kim K.H. Shcheynikov N. Wang Y. et al.SLC26A7 is a Cl− channel regulated by intracellular pH.J Biol Chem. 2005; 280: 6463-6470Crossref PubMed Scopus (108) Google Scholar, 21.Dorwart M.R. Shcheynikov N. Wang Y. et al.SLC26A9 is a Cl− channel regulated by the WNK kinases.J Physiol. 2007; 584: 333-345Crossref PubMed Scopus (92) Google Scholar Several SLC26 isoforms can transport oxalate, including SLC26A6 (PAT1), A7, A8, and A9.22.Xie Q. Welch R. Mercado A. et al.Molecular characterization of the murine Slc26a6 anion exchanger: functional comparison with Slc26a1.Am J Physiol Renal Physiol. 2002; 283: F826-F838Crossref PubMed Scopus (198) Google Scholar, 23.Jiang Z. Grichtchenko I.I. Boron W.F. et al.Specificity of anion exchange mediated by mouse Slc26a6.J Biol Chem. 2002; 277: 33963-33967Crossref PubMed Scopus (141) Google Scholar, 24.Clark J.S. Vandorpe D.H. Chernova M.N. et al.Species differences in Cl- affinity and in electrogenicity of SLC26A6-mediated oxalate/Cl- exchange correlate with the distinct human and mouse susceptibilities to nephrolithiasis.J Physiol. 2008; 586: 1291-1306Crossref PubMed Scopus (56) Google Scholar, 25.Freel R.W. Hatch M. Green M. et al.Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6 null mice.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G719-G728Crossref PubMed Scopus (128) Google Scholar, 26.Jiang Z. Asplin J.R. Evan A.P. et al.Calcium oxalate urolithiasis in mice lacking anion transporter Slc26a6.Nat Genet. 2006; 38: 474-478Crossref PubMed Scopus (228) Google Scholar SLC26 family members share a signature sequence on their C-terminal domain, which is referred to as sulfate transporter and anti-sigma factor domain and has an important role in transport function.1.Bissig M. Hagenbuch B. Stieger B. et al.Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.J Biol Chem. 1994; 269: 3017-3021PubMed Google Scholar, 2.Hastbacka J. de la Chapelle A. Mahtani M.M. et al.The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 3.Hoglund P. Haila S. Socha J. et al.Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhea.Nat Genet. 1996; 14: 316-319Crossref PubMed Scopus (322) Google Scholar, 4.Everett L.A. Glaser B. Beck J.C. et al.Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).Nat Genet. 1997; 17: 411-422Crossref PubMed Scopus (948) Google Scholar, 5.Zheng J. Shen W. He D.Z. et al.Prestin is the motor protein of cochlear outer hair cells.Nature. 2000; 405: 149-155Crossref PubMed Scopus (917) Google Scholar, 6.Lohi H. Kujala M. Kerkela E. et al.Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger.Genomics. 2000; 70: 102-112Crossref PubMed Scopus (189) Google Scholar, 7.Lohi H. Kujala M. Makela S. et al.Functional characterization of three novel tissue-specific anion exchangers SLC26A7, -A8, and -A9.J Biol Chem. 2002; 277: 14246-14254Crossref PubMed Scopus (182) Google Scholar, 8.Vincourt J.B. Jullien D. Amalric F. et al.Molecular and functional characterization of SLC26A11, a sodium-independent sulfate transporter from high endothelial venules.FASEB J. 2003; 17: 890-892Crossref PubMed Scopus (76) Google Scholar, 9.Romero M.F. Chang M.H. Plata C. et al.Physiology of electrogenic SLC26 paralogues.Novartis Found Symp. 2006; 273: 126-138Crossref PubMed Google Scholar, 10.Soleimani M. Xu J. SLC26 chloride/base exchangers in the kidney in health and disease.Semin Nephrol. 2006; 26: 375-385Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar At present, little is known about SLC26A11, other than that its mRNA is expressed in several tissues including the placenta, kidney, and venules, and can transport sulfate.8.Vincourt J.B. Jullien D. Amalric F. et al.Molecular and functional characterization of SLC26A11, a sodium-independent sulfate transporter from high endothelial venules.FASEB J. 2003; 17: 890-892Crossref PubMed Scopus (76) Google Scholar No studies have examined the cellular distribution and subcellular localization of SLC26A11 in the kidney or in any other tissues. Furthermore, the affinity of SLC26A11 for chloride transport or Cl−/HCO3− exchange, and its ability to facilitate any biological process remain unknown. Vacuolar H+-ATPase is expressed in the plasma membrane of specialized cells such as osteoclast and kidney collecting duct intercalated cells (review).27.Blake-Palmer K.G. Karet F.E. Cellular physiology of the renal H+ATPase.Curr Opin Nephrol Hypertens. 2009; 18: 433-438Crossref PubMed Scopus (18) Google Scholar,28.Brown D. Paunescu T.G. Breton S. et al.Regulation of the V-ATPase in kidney epithelial cells: dual role in acid-base homeostasis and vesicle trafficking.J Exp Biol. 2009; 212: 1762-1772Crossref PubMed Scopus (100) Google Scholar The secretion of acid through H+-ATPase into the lumen of the kidney collecting duct, and connecting, distal convoluted and proximal tubule has an essential role in systemic acid base homeostasis.29.Schwartz G.J. Barasch J. Al-Awqati Q. Plasticity of functional epithelial polarity.Nature. 1985; 318: 368-371Crossref PubMed Scopus (243) Google Scholar, 30.Alper S.L. Natale J. Gluck S. et al.Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase.Proc Natl Acad Sci USA. 1989; 86: 5429-5433Crossref PubMed Scopus (315) Google Scholar, 31.Valles P. Lapointe M.S. Wysocki J. et al.Kidney vacuolar H+ -ATPase: physiology and regulation.Semin Nephrol. 2006; 26: 361-374Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar It is well established that chloride has an important role in regulating the activity of V H+-ATPase expressed in the cellular and intracellular membranes.32.Carraro-Lacroix L.R. Lessa L.M. Fernandez R. et al.Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions.Braz J Med Biol Res. 2009; 42: 155-163Crossref PubMed Scopus (12) Google Scholar, 33.Malnic G. Geibel J.P. Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).J Membr Biol. 2000; 178: 115-125Crossref PubMed Scopus (25) Google Scholar, 34.Wagner C.A. Giebisch G. Lang F. et al.Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.Proc Natl Acad Sci USA. 1998; 95: 9665-9668Crossref PubMed Scopus (63) Google Scholar, 35.Tararthuch A.L. Fernandez R. Malnic G. Cl− and regulation of pH by MDCK-C11 cells.Braz J Med Biol Res. 2007; 40: 687-696Crossref PubMed Google Scholar The effect of chloride is likely mediated through two distinct mechanisms. The first is believed to be through the dissipation of a potential difference when chloride is secreted electrogenically, whereas the second is through a direct, specific stimulatory role that has been less well characterized.32.Carraro-Lacroix L.R. Lessa L.M. Fernandez R. et al.Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions.Braz J Med Biol Res. 2009; 42: 155-163Crossref PubMed Scopus (12) Google Scholar, 33.Malnic G. Geibel J.P. Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-).J Membr Biol. 2000; 178: 115-125Crossref PubMed Scopus (25) Google Scholar, 34.Wagner C.A. Giebisch G. Lang F. et al.Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.Proc Natl Acad Sci USA. 1998; 95: 9665-9668Crossref PubMed Scopus (63) Google Scholar, 35.Tararthuch A.L. Fernandez R. Malnic G. Cl− and regulation of pH by MDCK-C11 cells.Braz J Med Biol Res. 2007; 40: 687-696Crossref PubMed Google Scholar, 36.Hilden S.A. Johns C.A. Madias N.E. Cl(-)-dependent ATP-driven H+ transport in rabbit renal cortical endosomes.Am J Physiol. 1988; 255: F885-F897PubMed Google Scholar Our studies demonstrate that the expression of Slc26a11 in the kidney is exclusively limited to the collecting duct, with remarkable co-localization with H+-ATPase in intercalated cells. We further demonstrate that Slc26a11 mediates the transport of chloride and can facilitate acid extrusion through H+-ATPase. We propose that Slc26a11 is a functional partner of H+-ATPase and regulates acid secretion in the collecting duct. We first examined the distribution of Slc26a11 mRNA in mouse tissues. Figure 1a is a northern hybridization indicating the expression of Slc26a11 in multiple tissues, with abundant mRNA levels in the brain and kidney, followed by the distal colon. Next, reverse transcription-PCR experiments were performed on RNA isolated from various kidney zones to examine the zonal distribution of Slc26a11. The results demonstrated the expression of Slc26a11 in the cortex and medulla of mouse kidney (Figure 1b) as verified by sequencing of the single 1.2-kb band. Northern hybridization verified that Slc26a11 mRNA was abundantly expressed in the cortex and medulla (data not shown). This pattern of expression is distinct from Slc26a6 (PAT1), which is abundantly expressed in the cortex but is absent in the medulla, and Slc26a7 (PAT2), which is expressed in the medulla but not in the cortex.16.Wang Z. Wang T. Petrovic S. et al.Renal and intestine transport defects in Slc26a6-null mice.Am J Physiol Cell Physiol. 2005; 288: C957-C965Crossref PubMed Scopus (157) Google Scholar,17.Xu J. Song P. Nakamura S. et al.Deletion of the chloride transporter slc26a7 causes distal renal tubular acidosis and impairs gastric acid secretion.J Biol Chem. 2009; 284: 29470-29479Crossref PubMed Scopus (68) Google Scholar Mouse Slc26a11 has two distinct variants (GenBank accession numbers: AF345196 and BC132493) with the long variant having an additional 170 amino acids (aa) on its N-terminal end. In the next series of experiments, we examined the tissue distribution of long and short variants using variant-specific radiolabeled DNA probes (Materials and Methods). Figure 1c is a northern hybridization and shows that the long variant is predominantly expressed in the kidney and in several other epithelial tissues, including the brain. However, the short variant is predominantly expressed in the brain with lower levels in the kidney (Figure 1d). Given its abundant expression in the kidney and brain and its functional activity as an anion transporter (see below), we wish to designate Slc26a11 as KBAT (kidney brain anion transporter). Using an antibody generated against the mouse Slc26a11, a 72-kDa band was detected in microsomal membrane proteins from the renal medulla (Figure 2a, left panel). The labeling of the 72-kDa band was completely prevented with the pre-adsorbed immune serum (Figure 2a, left panel). To examine the specificity of the Slc26a11 antibody further, COS7 cells were transfected with the full-length Slc26a11 cDNA, labeled with Slc26a11 antibodies, and co-labeled with phalloidin-tetramethylrhodamine, a marker of the actin cytoskeleton. Images were taken on a Zeiss LSM510 confocal microscope (Zeiss, Thornwood, NY) and analyzed as before.17.Xu J. Song P. Nakamura S. et al.Deletion of the chloride transporter slc26a7 causes distal renal tubular acidosis and impairs gastric acid secretion.J Biol Chem. 2009; 284: 29470-29479Crossref PubMed Scopus (68) Google Scholar As indicated (Figure 2a, right panel), the Slc26a11 antibodies specifically label the plasma membrane of cells transfected with the Slc26a11 cDNA but not with the empty vector. To determine the cellular distribution and subcellular localization of KBAT, immunofluorescent staining with the purified immune serum was performed in the kidney. As shown in Figure 2bA–C, low-magnification images detected the expression of KBAT in a subset of cells in the collecting duct in the cortex, outer medulla, and inner medulla. As demonstrated in images with higher magnification, the labeling of KBAT in the cortex was detected on either the apical or the basolateral membrane domains in collecting duct cells (Figure 2cA), whereas the labeling of KBAT in the outer medullary collecting duct and inner medullary collecting duct was exclusively limited to the apical membrane (Figure 2cB and C). The labeling with KBAT antibodies was specific, as pre-adsorbed immune serum failed to detect any labeling in the kidney (Figure 2d). To determine the identity of Slc26a11-expressing cells, double-immunocytochemical staining with antibodies against Slc26a11 and aquaporin 2 (AQP2), which is exclusively expressed on the apical membrane of principal cells, was performed. As shown in Figure 3aA (the cortex, outer medulla, and inner medulla), KBAT (left) and AQP2 (right) clearly localized to two distinct cell populations in the cortical collecting duct (Figure 3aA), outer medullary collecting duct (Figure 3aB), and inner medullary collecting duct (Figure 3aC) (merged images in the middle), clearly demonstrating that cells expressing KBAT (Slc26a11) are distinct from principal cells. An obvious conclusion from these studies is that Slc26a11 (KBAT) is located in intercalated cells in the collecting duct. To examine this issue further, double-immunofluorescent labeling using KBAT and H+-ATPase antibodies was performed. As demonstrated, merged images display remarkably identical co-localization of KBAT with H+-ATPase along the length of the collecting duct (Figure 3bA–C). As shown, KBAT and H+-ATPase co-localize on the apical membrane of A-intercalated cells and on the basolateral membrane of B-intercalated cells in the cortical collecting duct (Figure 3bA). Some cells display co-localization of KBAT and H+-ATPase on the apical and basolateral membranes of the same cells (Figure 3bA). These latter cells most likely represent non-A-, non-B-intercalated cells, based on the reported H+-ATPase expression pattern. The co-localization of H+-ATPase and KBAT in the outer medullary collecting duct (Figure 3bB) and the initial portion of inner medullary collecting duct (Figure 3bC) was exclusively detected on the apical membrane of A-intercalated cells, consistent with the loss of non-A-intercalated cells in medullary collecting ducts. These results clearly demonstrate the co-localization of Slc26a11 (KBAT) with H+-ATPase in the collecting duct. To ascertain the functional identity of KBAT, COS7 cells were transiently transfected as described in the ‘Materials and Methods’ section. We first tested whether Slc26a11 has the ability to transport chloride. Toward this end, 24-well plates transfected with KBAT cDNA were used for 36Cl influx and efflux experiments as described in the ‘Materials and Methods’ section. As shown in Figure 4a, cells transiently transfected with Slc26a11 cDNA demonstrated increased uptake of 36Cl vs mock-transfected cells (P<0.02, n=6). The 10-min influx of 36Cl was significantly inhibited by 0.5mmol/l 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) in transfected cells (Figure 4a). We next examined the role of Slc26a11 in chloride efflux across the cell membrane in the absence or presence of external chloride. In the absence of extracellular chloride (all chloride salts were replaced with gluconate salts), Slc26a11-expressing cells demonstrated significant 36Cl efflux relative to mock-transfected cells, when an outwardly directed potassium gradient which causes depolarization (extracellular K=1mmol/l) was imposed, as shown in Figure 4b. In the presence of extracellular chloride (100mmol/l Cl−), the efflux of 36Cl was significantly increased in Slc26a11-transfected vs mock-transfected cells (P 2-fold higher relative to the absence of external chloride (Figure 4b). These results indicate that KBAT can function as an anion exchanger and as an electrogenic chloride-extruding pathway. To examine the effect of membrane potential alteration on KBAT, 36Cl efflux experiments were carried out under voltage-clamped conditions using valinomycin, a K+-specific ionophore, and a K+-rich solution and compared with a low K+ extracellular solution. The efflux of 36Cl was assayed in the presence or absence of chloride in the external solution. As shown in Figure 4c (left panel), in the absence of the external chloride but in the presence of valinomycin and K+-rich solution, 36Cl efflux in KBAT-transfected cells was significantly inhibited vs outwardly directed K gradient, and was not different relative to non-transfected cells (n=6, P>0.05). These results strongly suggest that the unidirectional chloride efflux (likely mediated through a conductive pathway) was regulated by membrane potential and is abrogated under voltage-clamped conditions. However, the efflux of 36Cl in the presence of external chloride was only partially inhibited under voltage-clamped conditions in KBAT-transfected cells (n=6, P<0.05 vs mock-transfected cells; Figure 4c, right panel), indicating that the KBAT-mediated anion exchange pathway is in a large part independent of membrane potential alteration. The 36Cl efflux in the presence or absence of extracellular chloride was significantly inhibited by 0.5mmol/l DIDS. Given the ability of KBAT to function as an anion exchanger (Figure 4b and c), we sought to determine whether KBAT could function in the Cl−/HCO3− exchange mode. Toward this end, COS7 cells were plated on coverslips and transiently transfected with KBAT cDNA. After 48h, cells were loaded with the pH-sensitive dye BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) and their intracellular pH (pHi) was monitored as described in the ‘Materials and Methods’ section. Our results demonstrated that in the presence of CO2/HCO3− in the solutions, sequential switching of the perfusate to chloride-free and chloride-containing solutions induced a significant pHi alteration in KBAT-transfected cells relative to mock-transfected cells (Figure 4d). The summation of six separate coverslips showed that in the presence of CO2/HCO3− in the media, the rate of intracellular alkalinization in response to chloride removal was 0.13±0.015 pH/min in KBAT-transfected and 0.04±0.007 in mock-transfected cells (P 0.05). In the presence of bicarbonate in the media, the total buffer capacity was 85.4±6.1mmol/l in transfected and 82.4±5.9mmol/l in non-transfected cells (n=3, P>0.05). Taken together, these results are consistent with KBAT functioning in the Cl−/HCO3− exchange mode. In the last series of experiments, we examined the effect of KBAT on H+-ATPase activity. Toward that end, cultured COS7 cells were plated on coverslips, transiently transfected with KBAT cDNA, and their pHi was examined 48h later by the pH-sensitive dye BCECF. To monitor H+ ATPase activity, cells were first acid loaded by NH4 pulse technique and then switched to a hypotonic Na-free solution. Under these conditions, pHi recovery from intracellular acidosis is exclusively mediated through H+-ATPase as determined by its sodium independence (Figure 5a) and its inhibition in the presence of bafilomycin (ref. 37.Bastani B. Purcell H. Hemken P. et al.Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat.J Clin Invest. 1991; 88: 126-136Crossref PubMed Scopus (181) Google Scholar and personal observation). As indicated, KBAT-transfected cells show an H+-ATPase activity, which was significantly increased relative to mock-transfected cells (Figure 5a). The results of six separate experiments demonstrated that the rate of H+-ATPase-mediated acid extrusion increased by ∼70% in KBAT-transfected cells (P<0.03, n=6) (Figure 5b). To examine the role of KBAT as a chloride transporter on H+-ATPase activity, cells were acid loaded and then assayed for pHi recovery in a chloride-depleted solution that in addition was sodium free (see the ‘Materials and Methods’ section). The results demonstrate that H+-ATPase activation by KBAT was almost abolished in chloride-depleted cells (Figure 5c, middle panel vs left panel). As indicated, mock-transfected cells showed moderate inhibition, whereas KBAT-transfected cells showed significant inhibition of H+-ATPase activity under chloride-depleted conditions. To further ascertain the role of KBAT on H+-ATPase activation, cells were monitored for pHi recovery from intracellular acidosis as described above in the presence of 0.5mmol/l DIDS. Our results demonstrate that the rate of Na-independent, bafilomycin-sensitive pHi recovery from acidosis (H+-ATPase activity) was significantly blunted in cells transfected with KBAT (Figure 5c, right panel vs left panel). As indicated, mock-transfected cells showed mild inhibition, whereas KBAT-transfected cells showed significant inhibition of their H+-ATPase activity by DIDS. To determine whether KBAT activation of H+-ATPase is mediated through its electrogenic transport of chloride, the experiments were repeated in the presence of high potassium in the perfusion solution (70mmol/l K+) and valinomycin at 10μmol/l (Materials and Methods). As shown in Figure 5c (right panel), the dissipation of membrane potential by increased external potassium and valinomycin only partially blocked the stimulation of H+-ATPase by KBAT. Taken together, these results strongly indicate that H+-ATPase activation of KBAT is dependent on intracellular chloride and is partially affected by alterations in membrane potential. The present studies identify Slc26a11 (KBAT) as a potential functional partner for V H+-ATPase in the kidney collecting duct. KBAT displays remarkable co-localization with H+-ATPase in the kidney collecting duct and is able to transport chloride when expressed in cultured cells. KBAT was able to function as an anion exchanger and as an electrogenic chloride-extruding pathway. KBAT enhanced the H+-ATPase-mediated acid extrusion by a chloride-dependent mechanism in cultured cells. The kidney collecting duct has an essential role in acid base transport and systemic pH homeostasis. This important function occurs by secretion of acid into the lumen, predominantly by vacuolar H+-ATPase.28.Brown
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