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• W2002671837 abstract "Cl-/HCO3- exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of HCO3- to the intracellular HCO3- binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/HCO3- exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/HCO3- exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/HCO3- exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/HCO3- exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/HCO3- exchange activity in AE1 mutants selectively impaired in Cl-/HCO3- exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/HCO3- exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/HCO3- exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO3- transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer. Cl-/HCO3- exchange activity mediated by the AE1 anion exchanger is reduced by carbonic anhydrase II (CA2) inhibition or by prevention of CA2 binding to the AE1 C-terminal cytoplasmic tail. This type of AE1 inhibition is thought to represent reduced metabolic channeling of HCO3- to the intracellular HCO3- binding site of AE1. To test the hypothesis that CA2 binding might itself allosterically activate AE1 in Xenopus oocytes, we compared Cl-/Cl- and Cl-/HCO3- exchange activities of AE1 polypeptides with truncation and missense mutations in the C-terminal tail. The distal renal tubular acidosis-associated AE1 901X mutant exhibited both Cl-/Cl- and Cl-/HCO3- exchange activities. In contrast, AE1 896X, 891X, and AE1 missense mutants in the CA2 binding site were inactive as Cl-/HCO3- exchangers despite exhibiting normal Cl-/Cl- exchange activities. Co-expression of CA2 enhanced wild-type AE1-mediated Cl-/HCO3- exchange, but not Cl-/Cl- exchange. CA2 co-expression could not rescue Cl-/HCO3- exchange activity in AE1 mutants selectively impaired in Cl-/HCO3- exchange. However, co-expression of transport-incompetent AE1 mutants with intact CA2 binding sites completely rescued Cl-/HCO3- exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl-/HCO3- exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO3- transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer. Na+-independent Cl-/HCO3- exchange contributes to the ability of cells to regulate their pH and volume, and to set the transmembrane electrochemical potentials for Cl- and HCO3- (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (169) Google Scholar). Na+-independent Cl-/HCO3- exchange is mediated by members of at least two gene families, SLC4 and SLC26 (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (169) Google Scholar, 2Kere J. Lohi H. Hoglund P. Am. J. Physiol. 1999; 276: G7-G16PubMed Google Scholar, 3Ko S.B. Shcheynikov N. Choi J.Y. Luo X. Ishibashi K. Thomas P.J. Kim J.Y. Kim K.H. Lee M.G. Naruse S. Muallem S. EMBO J. 2002; 21: 5662-5672Crossref PubMed Scopus (285) Google Scholar, 4Wang Z. Petrovic S. Mann E. Soleimani M. Am. J. Physiol. 2002; 282: G573-G579Crossref PubMed Scopus (228) Google Scholar). Within the SLC4 gene family, AE1, AE2, and AE3 (SLC4A1–3) (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (169) Google Scholar, 5Alper S.L. Darman R.B. Chernova M.N. Dahl N.K. J. Nephrol. 2002; 15: S41-S53PubMed Google Scholar, 6Sterling D. Casey J.R. Biochem. Cell Biol. 2002; 80: 483-497Crossref PubMed Scopus (80) Google Scholar) and perhaps also AE4 (SLC4A9) (7Tsuganezawa H. Kobayashi K. Iyori M. Araki T. Koizumi A. Watanabe S. Kaneko A. Fukao T. Monkawa T. Yoshida T. Kim D.K. Kanai Y. Endou H. Hayashi M. Saruta T. J. Biol. Chem. 2001; 276: 8180-8189Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) mediate Na+-independent Cl-/HCO3- exchange. These gene products encode polypeptides of tripartite structure, comprising an N-terminal cytoplasmic domain of 400–700 aa, 1The abbreviations used are: aa, amino acid(s); DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; wt, wild-type; BCECF, 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein; ANOVA, analysis of variance; eAE1, erythroid AE1; kAE1, kidney AE1; CA2, carbonic anhydrase II; dRTA, distal renal tubular acidosis. a transmembrane domain of 500 aa traversing the lipid bilayer 12–14 times, and a short C-terminal cytoplasmic domain. The N-terminal cytoplasmic domain links AE1 via ankyrin and the ERM protein 4.1R to the actin/spectrin cytoskeleton (3Ko S.B. Shcheynikov N. Choi J.Y. Luo X. Ishibashi K. Thomas P.J. Kim J.Y. Kim K.H. Lee M.G. Naruse S. Muallem S. EMBO J. 2002; 21: 5662-5672Crossref PubMed Scopus (285) Google Scholar, 6Sterling D. Casey J.R. Biochem. Cell Biol. 2002; 80: 483-497Crossref PubMed Scopus (80) Google Scholar, 8Low P.S. Biochim. Biophys. Acta. 1986; 864: 145-167Crossref PubMed Scopus (360) Google Scholar, 9Tanner M.J. Curr. Opin. Hematol. 2002; 9: 133-139Crossref PubMed Scopus (104) Google Scholar) and via protein 4.2 to the Rh antigen complex (10Bruce L.J. Ghosh S. King M.J. Layton D.M. Mawby W.J. Stewart G.W. Oldenborg P.A. Delaunay J. Tanner M.J. Blood. 2002; 100: 1878-1885Crossref PubMed Scopus (102) Google Scholar). The N-terminal cytoplasmic domain also provides a scaffold for enzymes of the glycolytic pathway, and binds denatured hemoglobin in pathological settings (8Low P.S. Biochim. Biophys. Acta. 1986; 864: 145-167Crossref PubMed Scopus (360) Google Scholar). The polytopic transmembrane domain (in the absence of nearly all the N-terminal cytoplasmic domain) suffices to mediate anion exchange by AE1 (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (169) Google Scholar, 5Alper S.L. Darman R.B. Chernova M.N. Dahl N.K. J. Nephrol. 2002; 15: S41-S53PubMed Google Scholar, 6Sterling D. Casey J.R. Biochem. Cell Biol. 2002; 80: 483-497Crossref PubMed Scopus (80) Google Scholar). Complete removal of the AE1 C-terminal cytoplasmic tail was shown some time ago to abolish AE1 function in Xenopus oocytes (11Chernova M.N. Humphreys B.D. Robinson D.H. Stuart-Tilley A.K. Garcia A.M. Brosius F.C. Alper S.L. Biochim. Biophys. Acta. 1997; 1329: 111-123Crossref PubMed Scopus (26) Google Scholar, 12Groves J.D. Tanner M.J. J. Biol. Chem. 1995; 270: 9097-9105Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), but recent years have seen a major re-evaluation of the functional role of this C-terminal tail. Erythroid AE1 (eAE1) is phosphorylated in intact red cells at aa Tyr-904 (13Brunati A.M. Bordin L. Clari G. James P. Quadroni M. Baritono E. Pinna L.A. Donella-Deana A. Blood. 2000; 96: 1550-1557Crossref PubMed Google Scholar) and at other tyrosine residues, but with still unknown consequence to anion transport rate or cytoskeletal association. The AE1 C-terminal tail associates in a yeast two-hybrid assay with the PDZ domain proteins syntenin and PICK-1 (14Cowan C.A. Yokoyama N. Bianchi L.M. Henkemeyer M. Fritzsch B. Neuron. 2000; 26: 417-430Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and the PMPV terminal sequence of AE1 has been proposed as a class II PDZ recognition sequence. However, p55, the only PDZ domain-containing protein of the red cell membrane identified to date, is thought to interact with AE1, if at all, indirectly via protein 4.1R (15Chishti A.H. Curr. Opin. Hematol. 1998; 5: 116-121Crossref PubMed Scopus (35) Google Scholar). The AE1 C-terminal tail also binds carbonic anhydrase II (CA2) (16Vince J.W. Reithmeier R.A. J. Biol. Chem. 1998; 273: 28430-28437Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Vince and Reithmeier (17Vince J.W. Reithmeier R.A. Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (162) Google Scholar) demonstrated that aa 886–890 of human eAE1 provided a core binding site for a positively charged region within the N-terminal 17 aa of CA2 (18Vince J.W. Carlsson U. Reithmeier R.A. Biochemistry. 2000; 39: 13344-13349Crossref PubMed Scopus (98) Google Scholar). Supuran et al. (19Scozzafava A. Supuran C.T. Bioorg. Med. Chem. Lett. 2002; 12: 1177-1180Crossref PubMed Scopus (62) Google Scholar) reported that peptides containing this core CA2 binding sequence could enhance CA2 activity in vitro. The important experiments of Sterling et al. (20Sterling D. Reithmeier R.A. Casey J.R. J. Biol. Chem. 2001; 27: 47886-47894Abstract Full Text Full Text PDF Scopus (315) Google Scholar) provided evidence that AE1 binding of functional CA2 is critical for AE1-mediated Cl-/HCO3- exchange activity transiently expressed in 293 cells, as well for activity of transfected AE2 and AE3. CA2 also binds to and appears to regulate activity of the SLC4A4 Na+-HCO3- cotransporter, NBCe1 (21Gross E. Pushkin A.M. Abuladze N. Fedotoff O. Kurtz I. J. Physiol. 2002; 544: 679-685Crossref PubMed Scopus (85) Google Scholar). Thus, CA2 binding may play an important general role in the function of SLC4 bicarbonate transporters, as well as for other pH-regulatory ion transporters (22Li X. Alvarez B. Casey J.R. Reithmeier R.A. Fliegel L. J. Biol. Chem. 2002; 277: 36085-36091Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). AE1 is so far unique among the electroneutral SLC4 anion exchanger genes in its association with Mendelian human diseases. Mutations in the AE1 gene cause autosomal dominant spherocytic anemia and Southeast Asian ovalocytosis, with no renal phenotype. A distinct set of mutations cause autosomal dominant or recessive forms of distal renal tubular acidosis (dRTA) without an erythroid phenotype (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (169) Google Scholar, 5Alper S.L. Darman R.B. Chernova M.N. Dahl N.K. J. Nephrol. 2002; 15: S41-S53PubMed Google Scholar). The eAE1 and kidney AE1 polypeptides (kAE1, expressed exclusively in type A intercalated cells of collecting duct) differ in the absence in kAE1 of the N-terminal 65 aa present in eAE1. Whereas the mutations associated with erythroid dyscrasias are distributed throughout the coding sequence of AE1, those associated to date with dRTA are restricted to the C-terminal transmembrane domain and to the short C-terminal cytoplasmic tail. Two families with dominant forms of dRTA have been reported to express distinct truncation mutations in the AE1 C-terminal tail (23Karet 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: 6337-6342Crossref PubMed Scopus (237) Google Scholar, 24Cheidde L. Vieira T.C Lima P.R. Saad S.T. Heilberg I.P. J. Am. Soc. Nephrol. 2002; 13: 575AGoogle Scholar). The AE1 901X mutation was associated with normal eAE1 polypeptide abundance and function in red cells, and exhibits wild-type (wt) Cl- influx in Xenopus oocytes (25Toye A.M. Bruce L.J. Unwin R.J. Wrong O. Tanner M.J. Blood. 2002; 99: 342-347Crossref PubMed Scopus (81) Google Scholar), properties shared with the dominant dRTA AE1 mutant R589H (26Bruce 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. Invest. 1997; 100: 1693-1707Crossref PubMed Scopus (313) Google Scholar, 27Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A.K. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Somers M.J.G. Seemanova E. Brouillette J. Brugnara C. Guay-Woodford L. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In contrast, kAE1 901X and the R589H mutants expressed in 293 cells failed to exit the endoplasmic reticulum (28Quilty J.A. Li J. Reithmeier R.A. Am. J. Physiol. 2002; 282: F810-F820Crossref PubMed Scopus (80) Google Scholar). The intracellular retention phenotype of kAE1 901X exhibited dominant negative properties in 293 cells (29Quilty J.A. Cordat E. Riethmeier R.A. Biochem. J. 2002; 368: 895-903Crossref PubMed Google Scholar), but these phenotypes differed in polarized epithelial cells (30Devonald M.A. Ihrke G. Smith A.N. Karet F.E. J. Am. Soc. Nephrol. 2002; 13: 57AGoogle Scholar). 2S. L. Alper and D. Prabakaran, unpublished results. The CA2 binding site of the AE1 C-terminal tail lies close to the terminus of the truncated dRTA mutant AE1 R901X, but the consequence of this truncation to Cl-/HCO3- exchange activity has not been tested. The idea of a bicarbonate transport metabolon (31Reithmeier R.A. Blood Cells Mol. Dis. 2001; 27: 35-39Crossref PubMed Scopus (79) Google Scholar) is an important and attractive one, but the mechanism by which CA2 stimulates HCO3- transport by SLC4 anion transport proteins remains uncertain. Bound CA2 might enhance AE1 activity by channeling HCO3- to or away from the cytoplasmic anion translocation pathway binding site of AE1 (20Sterling D. Reithmeier R.A. Casey J.R. J. Biol. Chem. 2001; 27: 47886-47894Abstract Full Text Full Text PDF Scopus (315) Google Scholar, 31Reithmeier R.A. Blood Cells Mol. Dis. 2001; 27: 35-39Crossref PubMed Scopus (79) Google Scholar). However, another AE1-binding protein, glycophorin A, is thought to alter the conformational state of AE1 to accelerate its biosynthetic delivery to the cell surface (32Groves J.D. Tanner M.J. J. Biol. Chem. 1992; 267: 22163-22170Abstract Full Text PDF PubMed Google Scholar), and perhaps also to increase the anion transport rate of AE1 at the cell surface (33Bruce L.J. Groves J.D. Okubo Y. Thilaganathan B. Tanner M.J. Blood. 1994; 84: 916-922Crossref PubMed Google Scholar). The possibility of a similar conformational role for CA2 binding on AE1 function has not been tested. Prompted by these and other considerations, we tested the hypothesis that CA2 binding mediates not only metabolic channeling of substrate, but also might allosterically up-regulate AE1 activity. To do so, we compared in Xenopus oocytes the consequences of various AE1 C-terminal tail mutations to kAE1-mediated Cl-/Cl- and Cl-/HCO3- exchange activities in the absence and presence of co-expressed CA2. The results of these experiments, although not supporting allosteric regulation of AE1 by CA2, enhance understanding of the role of CA2 activity in AE1 function and enlarge the extent of a functionally defined core CA2 binding site. In providing a new constraint on the molecular distance across which CA2 can activate AE1, the data also reveal a possible role for inter-protomeric interactions in AE1-mediated Cl-/HCO3- exchange. Finally, the data provide additional strong evidence that an AE1 monomer encompasses an intact transbilayer anion translocation pathway. cDNAs and Mutagenesis—cDNAs encoding human kAE1 (27Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A.K. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Somers M.J.G. Seemanova E. Brouillette J. Brugnara C. Guay-Woodford L. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 34Tanphaichitr V.S. Sumboonnanonda A. Ideguchi H. Shayakul C. Brugnara C. Takao M. Veerakul G. Alper S.L. J. Clin. Invest. 1998; 102: 2173-2179Crossref PubMed Scopus (158) Google Scholar), human eAE1 SAO (Southeast Asian Ovalocytosis) (11Chernova M.N. Humphreys B.D. Robinson D.H. Stuart-Tilley A.K. Garcia A.M. Brosius F.C. Alper S.L. Biochim. Biophys. Acta. 1997; 1329: 111-123Crossref PubMed Scopus (26) Google Scholar, 35Schofield A.E. Reardon D.M. Tanner M.J. Nature. 1992; 355: 836-838Crossref PubMed Scopus (147) Google Scholar), and mouse eAE1 E699Q (36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar) were previously described. cDNAs encoding human CA2 (gift of W. Sly, St. Louis University, St. Louis, MO) and the CA2 mutants V143Y (37Fierke C.A. Calderone T.L. Krebs J.F. Biochemistry. 1991; 30: 11054-11063Crossref PubMed Scopus (125) Google Scholar) (gift of C. Fierke, University of Michigan, Ann Arbor, MI) and ΔN17/C206S cDNA (38Aronsson G. Martensson L. Carlsson U. Jonsson B. Biochemistry. 1995; 34: 2153-2162Crossref PubMed Scopus (41) Google Scholar) (gift of U. Carlsson, University of Linkoping, Linkoping, Sweden) were subcloned into the oocyte expression vector pXT7. Mutations in the C-terminal cytoplasmic tail of kAE1were generated by four-primer polymerase chain reaction (PCR) as previously described (36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar). Locations of introduced termination mutations are indicated as X in Fig. 1. Location of the “core” CA2 binding sequence LDADD (17Vince J.W. Reithmeier R.A. Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (162) Google Scholar) is boxed and shaded in Fig. 1. This wild-type sequence was mutated to the active variant LDPDD, and to the inactive variants ADADD and LDAAA (17Vince J.W. Reithmeier R.A. Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (162) Google Scholar). Primer sequences are available on request. Heterologous Protein Expression in Xenopus Oocytes—cRNAs were transcribed from appropriately linearized templates with the Megascript kit (Ambion, Woodlands, TX). Xenopus oocytes were harvested and defolliculated as described (36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar, 39Stewart A.K. Chernova M.N. Shmukler B.E. Wilhelm S. Alper S.L. J. Gen. Physiol. 2002; 120: 707-722Crossref PubMed Scopus (63) Google Scholar), then injected with 50 nl of cRNA solution containing (unless otherwise indicated) 5 ng of wild-type or mutant kAE1 cRNA and/or 1 ng wild-type or mutant CA2 cRNA. cRNA-injected oocytes were incubated at 19 °C in ND96 solution containing (in mm): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 2.5 sodium pyruvate, and 100 μg/ml gentamicin. Oocytes were studied 2–6 days following cRNA injection. Unidirectional 36Cl - Influx and Efflux Assays— 36Cl- influx was measured as previously described (11Chernova M.N. Humphreys B.D. Robinson D.H. Stuart-Tilley A.K. Garcia A.M. Brosius F.C. Alper S.L. Biochim. Biophys. Acta. 1997; 1329: 111-123Crossref PubMed Scopus (26) Google Scholar, 36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar, 40Zhang Y. Chernova M. Stuart-Tilley A. Jiang L. Alper S.L. J. Biol. Chem. 1996; 271: 5741-5749Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) in ND96 solution lacking sodium pyruvate and gentamicin, and containing 3 μCi/150 μl assay volume. Influx was linear over 15 min. 36Cl- efflux was measured essentially as described (36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar, 39Stewart A.K. Chernova M.N. Shmukler B.E. Wilhelm S. Alper S.L. J. Gen. Physiol. 2002; 120: 707-722Crossref PubMed Scopus (63) Google Scholar). Oocytes were injected with 10,000–25,000 36Cl-, allowed to recover for 10 min in Cl--free medium, then transferred individually into 1 ml of medium containing Cl- (ND-96) or 5% CO2/24 mmNaHCO3-/72 mm sodium gluconate. These 36Cl--loaded oocytes began the 36Cl- efflux assay with calculated [Cl-]i 26 mm higher than the endogenous value of ∼35 mm (36Chernova M.N. Jiang L. Vandorpe D.H. Hand M. Crest M. Strange K. Alper S.L. J. Gen. Physiol. 1997; 109: 345-360Crossref PubMed Scopus (83) Google Scholar) in the oocytes studied in the 36Cl- influx assay. 950 μl of medium was collected at 3-min intervals and replaced with fresh medium of desired composition. All assays included a final efflux period in the presence of the anion transport inhibitor, DIDS (200 μm). Oocytes exhibiting <50% inhibition of 36Cl- efflux by DIDS were considered leaky, and excluded from analysis. These comprised fewer than 2% of the total studied. All flux experiments were performed with oocytes from at least two frogs. Cl-/HCO3- Exchange Measurement—Oocytes previously injected with water or with kAE1 cRNA were loaded with 5 μm BCECF-AM for 30 min, and mounted in a 0.8-ml superfusion chamber on a microscope stage. Cl-/HCO3- exchange was measured by BCECF fluorescence ratio imaging of oocyte pHi changes during removal and restoration of perfusate Cl- (72 mm) in the presence of 5% CO2, 24 mmNaHCO3-, with gluconate as substituting anion (41Jiang L.W. Chernova M.N. Alper S.L. Am. J. Physiol. 1997; 272: C191-C202Crossref PubMed Google Scholar). Data acquisition in early experiments was with Image-1 FL and analysis with Image-1 (Universal Imaging, West Chester, PA), but subsequent data acquisition and analysis used Metamorph (Universal Imaging). Initial rates of dpHi/dt following solution changes of superfusate were measured by least squares linear fit of initial slopes. Initial pHi in the presence of CO2/HCO3- was indistinguishable among all groups of oocytes reported this paper. Thus, dpHi/dt was proportional to proton equivalent flux. All experimental groups with >6 oocytes derived from at least two frogs. Groups of 6 oocytes were either from two frogs, or represented oocytes from one frog evaluated on two successive days. Immunoblot—Groups of 5–10 individually defolliculated oocytes, or an equal number of folliculated oocytes within a cluster resected from an ovarian fragment, were homogenized on ice in 140 mm NaCl, 10 mm sodium phosphate, pH 7.4, and immediately solubilized in an equal volume of 2× Laemmli SDS load buffer. Protein (normalized to starting oocyte number) was subjected to SDS-PAGE on a 4–20% gradient gel, blotted to nitrocellulose, and probed with rabbit antibody to bovine erythroid carbonic anhydrase 2 or to human erythroid carbonic anhydrase 1 (both from Polysciences, Warrenton, PA), followed by peroxidase-coupled anti-Ig (Jackson ImmunoResearch, West Grove, PA) and development by enhanced chemiluminescence (PerkinElmer Life Sciences). Functional Effects of Sequential Truncation of the kAE1 C-terminal Cytoplasmic Tail—We exploited the ability to compare Cl-/Cl- and Cl-/HCO3- exchange activities in Xenopus oocytes to test two initial hypotheses. The first hypothesis proposed that the dRTA mutation 901X might impair selectively Cl-/HCO3- exchange activity while preserving Cl-/Cl- exchange activity. The second hypothesis proposed that CA2 binding might serve not only to provide a locally elevated concentration of transport substrate, but might also control anion transport rate by inducing or stabilizing an AE1 conformation favoring or required for transport. Such a conformational change might be evident in an effect of CA2 binding on kAE1-mediated Cl-/Cl- exchange. Fig. 1 depicts the truncated polypeptides examined in this study. Fig. 2 compares the effects on kAE1-mediated Cl-/Cl- and Cl-/HCO3- exchange activities of progressive truncation of the C-terminal cytoplasmic tail of kAE1. Fig. 2A shows that the dRTA mutant 901X lacking the C-terminal 11 aa of kAE1 exhibits wt Cl- influx into Xenopus oocytes, as reported previously (25Toye A.M. Bruce L.J. Unwin R.J. Wrong O. Tanner M.J. Blood. 2002; 99: 342-347Crossref PubMed Scopus (81) Google Scholar). Cl- influx mediated by kAE1 901X did not differ from wt whether 1, 5, or 10 ng of cRNA was injected, did not exhibit a dominant negative phenotype when co-expressed with wt AE1, and was normally stimulated by co-expressed glycophorin A (25Toye A.M. Bruce L.J. Unwin R.J. Wrong O. Tanner M.J. Blood. 2002; 99: 342-347Crossref PubMed Scopus (81) Google Scholar) (data not shown). Engineered truncations lacking 16 aa (kAE1 896X) and 21 aa (891X) also mediated wt rates of Cl- influx. However, truncation of 27 aa (885X) or of 30 aa (882X) abolished Cl- influx activity. kAE1 891X and 896X similarly displayed wt Cl-/Cl- exchange activity measured as 36Cl- efflux (Fig. 2B). The dRTA mutant kAE1 901X also preserved Cl-/Cl- exchange activity, although at reduced rates in these experiments. Cl-/HCO3- exchange mediated by kAE1 901X was only slightly (but statistically significantly) reduced compared with wt kAE1 (Fig. 2, C and D). However, further truncation of kAE1 abolished Cl-/HCO3- exchange. Thus, despite their wt rates of Cl-/Cl- exchange (Fig. 2, A and B), the kAE1 truncation mutants 896X and 891X were devoid of Cl-/HCO3- exchange activity (Fig. 2, C and D). This property was unexpected, because both of these truncation mutants preserve the in vitro defined LDAAA core AE1 binding site for CA2 comprising AE1 residues 886–890. The observed specific loss of Cl-/HCO3- exchange activity suggests that the CA2 binding site of AE1 as delimited by functional studies must be more extensive than the “LDADD” core site defined by CA2 binding to AE1 fusion protein (17Vince J.W. Reithmeier R.A. Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (162) Google Scholar). The retention of Cl-/Cl- exchange activity by these two kAE1 mutants incapable of Cl-/HCO3- exchange activity does not support the hypothesis that CA2 binding changes AE1 conformation in a manner that can regulate transport rate of non-bicarbonate anion. CA2 Coexpression with kAE1 Stimulates Cl-/HCO3- but Not Cl - /Cl - Exchange—Co-expression of wild-type CA2 with eAE1 in 293 cells does not accelerate Cl-/HCO3- exchange rates (20Sterling D. Reithmeier R.A. Casey J.R. J. Biol. Chem. 2001; 27: 47886-47894Abstract Full Text Full Text PDF Scopus (315) Google Scholar). Coexpression of CA2 with kAE1 in Xenopus oocytes offers the opportunity to compare effects of CA2 on kAE1-mediated Cl-/Cl- and Cl-/HCO3- exchange activities. Fig. 3A shows that CA2 does not itself increase 36Cl- influx into water-injected oocytes. Moreover, CA2 co-expression with kAE1 even at nominal 3–4-fold molar excess of cRNA (10 ng) did not enhance kAE1-mediated 36Cl- influx. kAE1-mediated 36Cl- efflux into 24 mmHCO3- and into ND-96 were compared in the same oocytes (Fig. 3B). Efflux required permeant extracellular anion (isethionate supported only minimal efflux) and was inhibited by DIDS. As shown in Fig. 3C, CA2 coexpression led to a slight but statistically significant reduction in 36Cl- efflux into extracellular Cl-, and inhibited by 50% 36Cl- efflux into Cl--free CO2/HCO3- medium. These results suggest increased generation of HCO3- by CA2 near the intracellular face of the plasma membrane, where it can compete for efflux by kAE1 with intracellular 36Cl-. In contrast, CA2 co-expression enhanced Cl-/HCO3- exchange activity mediated by wt kAE1 as well as that by the dRTA mutant kAE1 901X. However, CA2 co-expression with the more extensively truncated kAE1 mutants 896X and 891X did not lead to rescue of their Cl-/HCO3- exchange activity (Fig. 3D). kAE1 Missense Mutations Which Abrogate CA2 Binding Selectively Abolish Cl-/HCO3- Exchange but Maintain Cl - /Cl - Exchange—Selected missense mutations in the core CA2 binding site of the AE1 C-terminal cytoplasmic tail have been shown to abrogate binding of CA2 (17Vince J.W. Reithmeier R.A. Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (162) Google Scholar). Mutation of the core binding sequence LDAAA to ADADD or to LDAAA abolished CA2 binding to AE1 fusion protein, whereas conversion to LDPDD retained binding. Interestingly, all three of these missense mutant polypeptides retained wild-type rates of Cl-/Cl- exchange measured as 36Cl- influx (Fig. 4A). Cl-/Cl- exchange measured as 36Cl- efflux was also preserved in all these mutants, although not quite at wild-type levels (Fig. 4B). In contrast, Cl-/HCO3- exchange activities correlated with previously reported CA2 binding properties. Whereas the mutants LDAAA and ADADD exhibited no Cl-/HCO3- exchange activity and were unaffected by coexpression of CA2, the mutant LDPDD retained near-wt Cl-/HCO3- exchange activity, which was further increased by co-expression of CA2 (Fig. 4, C and D). A Transport-incompetent AE1 Mutant with an Intact CA2 Binding Site Rescues the Cl-/HCO3- Exchange Activity of a kAE1 Missense Mutant Deficient in CA2 Binding—The above experiments revealed that kAE1 required an intact “functional CA2 binding site” to respond to CA2 co-expression with enhanced Cl-/HCO3- exchange activity. However, neither CA2 co-expression nor integrity of the CA2 binding" @default.
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• W2002671837 title "Deficient <mml:math xmlns:mml=http://www.w3.org/1998/Math/MathML altimg=si1.gif><mml:msubsup><mml:mi>HCO</mml:mi><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mo>-</mml:mo></mml:msubsup></mml:math> Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer" @default.
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