The role of carbonic anhydrases in renal physiology

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2. CA is expressed in most segments of the kidney. CAII and CAIV predominate in human and rabbit kidneys; in rodent kidneys, CAXII, and CAXIV are also present. CAIX is expressed by renal cell carcinoma (RCC). Most of these isoforms, except for rodent CAIV, have high turnover rates. CAII is a cytoplasmic enzyme, whereas the others are membrane-associated; CAIV is anchored by glycosylphosphatidylinositol linkage. Membrane polarity is apical for CAXIV, basolateral for CAXII, and apical and basolateral for CAIV. Luminal membrane CAs facilitate the dehydration of carbonic acid (H2CO3) that is formed when secreted protons combine with filtered bicarbonate. Basolateral CA enhances the efflux of bicarbonate via dehydration of H2CO3. CAII and CAIV can associate with bicarbonate transporters (e.g., AE1, kNBC1, NBC3, and SCL26A6), and proton antiporter, NHE1 in a membrane protein complex called a transport metabolon. CAXII and CAXIV may also be associated with transporters in normal kidney and CAIX in RCCs. The multiplicity of CAs implicates their importance in acid–base and other solute transport along the nephron. For example, CAII on the cytoplasmic face and CAIV on the extracellular surface provide the 'push' and 'pull' for bicarbonate transport by supplying and dissipating substrate respectively. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2. CA is expressed in most segments of the kidney. CAII and CAIV predominate in human and rabbit kidneys; in rodent kidneys, CAXII, and CAXIV are also present. CAIX is expressed by renal cell carcinoma (RCC). Most of these isoforms, except for rodent CAIV, have high turnover rates. CAII is a cytoplasmic enzyme, whereas the others are membrane-associated; CAIV is anchored by glycosylphosphatidylinositol linkage. Membrane polarity is apical for CAXIV, basolateral for CAXII, and apical and basolateral for CAIV. Luminal membrane CAs facilitate the dehydration of carbonic acid (H2CO3) that is formed when secreted protons combine with filtered bicarbonate. Basolateral CA enhances the efflux of bicarbonate via dehydration of H2CO3. CAII and CAIV can associate with bicarbonate transporters (e.g., AE1, kNBC1, NBC3, and SCL26A6), and proton antiporter, NHE1 in a membrane protein complex called a transport metabolon. CAXII and CAXIV may also be associated with transporters in normal kidney and CAIX in RCCs. The multiplicity of CAs implicates their importance in acid–base and other solute transport along the nephron. For example, CAII on the cytoplasmic face and CAIV on the extracellular surface provide the 'push' and 'pull' for bicarbonate transport by supplying and dissipating substrate respectively. Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible hydration of CO2 according to the reaction:H++HCO3−↔H2CO3↔CACO2+H2O(1) CO2 gas dissolves in water and is in equilibrium with the acid H2CO3. The Henderson–Hasselbalch equation relates pH, HCO3− concentration, and partial pressure of CO2 gas in physiologic solutions:pH=6.1+log([HCO3−]/[0.03*pCO2])(2) The uncatalyzed hydration of CO2 is relatively slow, whereas the turnover number for CAII is of the order of 106 s−1.1.Khalifah R.G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C.J Biol Chem. 1971; 246: 2561-2573Abstract Full Text PDF PubMed Google Scholar,2.Maren T.H. Carbonic anhydrase: chemistry, physiology and inhibition.Physiol Rev. 1967; 47: 595-781Crossref PubMed Scopus (1393) Google Scholar At present there are 15 known isoforms of CA, which differ in kinetic properties, susceptibility to inhibitors, and specific tissue distribution. From the reactions 1 and 2 above, it is logical to assume that CA can facilitate renal acidification, because the concentrations of CO2 and HCO3− are interdependent. In addition, CA can modulate acid–base transport in three other ways. First, CA can make dissolved HCO3− available for rapid conversion to CO2, thereby increasing the amount of transported CO2 by the amount of HCO3− present in solution. Second, CA can mobilize HCO3− diffusion for CO2 transport, in a process known as facilitated diffusion, whereby there is HCO3− diffusion and simultaneous H+ transport in the presence of a mobile buffer, such as phosphate or albumin.3.Gros G. Moll W. Facilitated diffusion of CO2 across albumin solutions.J Gen Physiol. 1974; 64: 356-371Crossref PubMed Scopus (42) Google Scholar,4.Gros G. Moll W. Hoppe H. Gros H. Proton transport by phosphate diffusion – A mechanism of facilitated CO2 transfer.J Gen Physiol. 1976; 67: 773-790Crossref PubMed Scopus (55) Google Scholar For example, at pH 7.4 there are 20 times more HCO3− ions as CO2 molecules, so that CO2 diffusion may be increased by 20-fold.4.Gros G. Moll W. Hoppe H. Gros H. Proton transport by phosphate diffusion – A mechanism of facilitated CO2 transfer.J Gen Physiol. 1976; 67: 773-790Crossref PubMed Scopus (55) Google Scholar,5.Enns T. Facilitation by carbonic anhydrase of carbon dioxide transport.Science. 1967; 155: 44-47Crossref PubMed Scopus (118) Google Scholar Third, Enns5.Enns T. Facilitation by carbonic anhydrase of carbon dioxide transport.Science. 1967; 155: 44-47Crossref PubMed Scopus (118) Google Scholar showed that CA alone, in the absence of HCO3− ions, (pH 5.2), directly enhances transport, perhaps by acting as a carrier for CO2. CAII accounts for >95% of CA activity in the kidney and resides in the cytosol. In non-rodent species (e.g. human, rabbit, and bovine) most of the remaining ∼5% of renal CA is membrane-associated and comprised of CAIV and CAXII.6.Wistrand P.J. Knuuttila K.-G. Renal membrane-bound carbonic anhydrase. Purification and properties.Kidney Int. 1989; 35: 851-859Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 7.Brion L.P. Zavilowitz B.J. Suarez C. Schwartz G.J. Metabolic acidosis stimulates carbonic anhydrase activity in rabbit proximal tubule and medullary collecting duct.Am J Physiol. 1994; 266: F185-F195PubMed Google Scholar, 8.McKinley D.N. Whitney P.L. Particulate carbonic anhydrase in homogenates of human kidney.Biochim Biophys Acta. 1976; 445: 780-790Crossref PubMed Scopus (59) Google Scholar In addition to CAIV and CAXII, CAXIV, and CAXV are expressed in the kidney of rodent species.9.Mori K. Ogawa Y. Ebihara K. et al.Isolation and characterization of CAXIV, a novel membrane-bound carbonic anhydrase from mouse kidney.J Biol Chem. 1999; 274: 15701-15705Crossref PubMed Scopus (111) Google Scholar, 10.Fujikawa-Adachi K. Nishimori I. Taguchi T. Onishi S. Human carbonic anhydrase XIV (CA14): cDNA cloning, mRNA expression, and mapping to chromosome 1.Genomics. 1999; 61: 74-81Crossref PubMed Scopus (105) Google Scholar, 11.Fujikawa-Adachi K. Nishimori I. Taguchi T. et al.cDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI.Biochim Biophys Acta. 1999; 1431: 518-524Crossref PubMed Scopus (22) Google Scholar, 12.Hilvo M. Tolvanen M. Clark A. et al.Characterization of CAXV, a new GPI-anchored form of carbonic anhydrase.Biochem J. 2005; 392: 83-92Crossref PubMed Scopus (136) Google Scholar Although CAIX is not expressed in normal kidney, it is commonly found in neoplasms arising from renal tissue, particularly renal clear-cell carcinoma.13.Ivanov S.V. Kuzmin I. Wei M.-H. et al.Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel–Lindau transgenes.Proc Natl Acad Sci USA. 1998; 95: 12596-12601Crossref PubMed Scopus (327) Google Scholar In the following sections we will examine the function of CA isoforms in acid/base physiology of the kidney by defining the following parameters for each of the renal CA isoforms: (1) expression along the nephron segments (cell types), (2) subcellular localization, and (3) functional associations with acid/base transporters. Studies within the last 10 years have demonstrated that CAs associate with acid/base transporters to form H+/HCO3− transport metabolons.14.McMurtrie H.L. Cleary H.J. Alvarez B.V. et al.The bicarbonate transport metabolon.J Enzyme Inhib Med Chem. 2004; 19: 231-236Crossref PubMed Scopus (113) Google Scholar Association of CAs with transporters has been shown to markedly enhance H+/HCO3− transport, and thus via mass action CAs provide the 'push' and 'pull' for H+/HCO3− transport by accumulating or dissipating substrate. To date 15 CA isoforms have been identified of which twelve are catalytically active; CAs VIII, X, and XI are CA-related proteins that are not catalytically active owing to the absence of one or more of the conserved histidine residues that are required for coordination of Zn, which is essential for CA enzyme catalysis.11.Fujikawa-Adachi K. Nishimori I. Taguchi T. et al.cDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI.Biochim Biophys Acta. 1999; 1431: 518-524Crossref PubMed Scopus (22) Google Scholar,15.Okamoto N. Fujikawa-Adachi K. Nishimori I. et al.cDNA sequence of human carbonic anhydrase-related protein, CA-RP X: mRNA expressions of CA-RP X and XI in human brain.Biochim Biophys Acta. 2001; 1518: 311-316Crossref PubMed Scopus (28) Google Scholar CAs can be divided into three groups based on their domain structure that is comprised of a CA catalytic domain along with other structural features that influence cellular localization (Figure 1). The cytosolic CA isoforms for which CAII serves as a prototype, also include CAs I, III, and VII, which are comprised of a CA domain encompassing the enzyme active site and the three conserved histidine residues found in all catalytically active CA isoforms. The CAV isoforms are expressed in mitochondria owing to the inclusion of a 33 amino-acid classical mitochondrial leader sequence in the 317 amino-acid precursor peptide containing a CA domain. The CAV isoform includes two homologs, CAVA and CAVB that differ in their tissue distributions: CAVB is expressed in most tissues, whereas CAVA expression is restricted to liver, skeletal muscle, and kidney.16.Shah G.N. Hewett-Emmett D. Grubb J.H. et al.Mitochondrial carbonic anhydrase CAVB: differences in tissue distribution and pattern of evolution from those of CAVA suggest distinct physiological roles.Proc Natl Acad Sci USA. 2000; 97: 1677-1682Crossref PubMed Scopus (68) Google Scholar Addition of a signal sequence to a CA domain and a C-terminal extension that is rich in hydrophilic amino acid produces CAVI, the lone secreted isoform which is expressed in the salivary gland of a number of mammalian species.17.Fernley R.T. Wright R.D. Coghlan J.P. Complete amino acid sequence of ovine salivary carbonic anhydrase.Biochemistry. 1988; 27: 2815-2820Crossref PubMed Scopus (43) Google Scholar,18.Leinonen J. Parkkila S. Kaunisto K. et al.Secretion of carbonic anhydrase isoenzyme VI (CAVI) from human and rat lingual serous von Ebner's glands.J Histochem Cytochem. 2001; 49: 657-662Crossref PubMed Scopus (53) Google Scholar There are now five membrane-associated CA isoforms with the recent addition of CAXV to this group,12.Hilvo M. Tolvanen M. Clark A. et al.Characterization of CAXV, a new GPI-anchored form of carbonic anhydrase.Biochem J. 2005; 392: 83-92Crossref PubMed Scopus (136) Google Scholar three of which (CAs IX, XII, and XIV) are expressed as single-pass transmembrane proteins.9.Mori K. Ogawa Y. Ebihara K. et al.Isolation and characterization of CAXIV, a novel membrane-bound carbonic anhydrase from mouse kidney.J Biol Chem. 1999; 274: 15701-15705Crossref PubMed Scopus (111) Google Scholar, 19.Pastorek J. Pastorekova S. Callebaut I. et al.Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.Oncogene. 1994; 9: 2877-2888PubMed Google Scholar, 20.Tureci O. Sahin U. Vollmar E. et al.Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers.Proc Natl Acad Sci USA. 1998; 95: 7608-7613Crossref PubMed Scopus (299) Google Scholar, 21.Schwartz G.J. Kittelberger A.M. Watkins R.H. O'Reilly M.A. Carbonic anhydrase XII mRNA encodes a hydratase that is differentially expressed along the rabbit nephron.Am J Physiol. 2003; 284: F399-F410PubMed Google Scholar In addition to a conserved catalytic domain found in all CA isoforms, CAIX contains an N-terminal proteoglycan-like domain that may function in cell adhesion.22.Zavada J. Zavadova Z. Pastorek J. et al.Human tumour-associated cell adhesion protein MN/CAIX: identification of M75 epitope and of the region mediating cell adhesion.Br J Cancer. 2000; 82: 1808-1813Crossref PubMed Scopus (137) Google Scholar CAIV and the closely related CAXV,12.Hilvo M. Tolvanen M. Clark A. et al.Characterization of CAXV, a new GPI-anchored form of carbonic anhydrase.Biochem J. 2005; 392: 83-92Crossref PubMed Scopus (136) Google Scholar are tethered to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol lipid anchor (GPI anchor) that typically results in the luminal or apical surface expression of the protein in polarized epithelial cells.23.Waheed A. Zhu X.L. Sly W.S. Membrane-associated carbonic anhydrase from rat lung: purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.J Biol Chem. 1992; 267: 3308-3311Abstract Full Text PDF PubMed Google Scholar, 24.Zhu X.L. Sly W.S. Carbonic anhydrase IV from human lung: purification, characterization, and comparison with membrane carbonic anhydrase from human kidney.J Biol Chem. 1990; 265: 8795-8801Abstract Full Text PDF PubMed Google Scholar, 25.Powell S.K. Cunningham B.A. Edelman G.M. Rodriguez-Boulan E. Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.Nature. 1991; 353: 76-77Crossref PubMed Scopus (84) Google Scholar However, recent studies in our laboratory have revealed an exception to this rule for CAIV expression in proximal tubule epithelial cells, as is discussed in detail below. The α class of CAs comprises zinc-containing metalloenzymes that catalyze the reversible hydration of CO2 according to the reaction (3):CO2+H2O↔CAH2CO3↔H++HCO3−(3) CO2 hydration occurs in two distinct steps26.Silverman D.N. Lindskog S. The catalytic mechanism of carbonic anhydrases: implications of rate-limited protolysis of water.Acc Chem Res. 1988; 21: 30-36Crossref Scopus (670) Google Scholar as shown below.E−Zn−OH−+CO2↔E−Zn−HCO3−+H2O↔E−Zn−OH2+HCO3−(4) First a nucleophilic attack by the zinc-bound hydroxide occurs at the carbonyl carbon of CO2 to form zinc-bound bicarbonate. For the prototype CAII enzyme the second-order rate constant for CO2 hydration (Kcat/Km) approaches the diffusion control limit. The second step regenerates the active zinc hydroxide species, when a proton is transferred from zinc-bound water to a solvent buffer molecule in two stages via the His64.His(64)−E−Zn−OH2↔H+−His(64)−E−Zn−OH−+B↔His(64)−E−Zn−OH−+B−H+(5) In stage 1 there is an intra-molecular transfer of a proton from zinc-bound water to a protein side chain with a pKa of 7, then an inter-molecular transfer of the proton to solvent buffer molecule. In reaction (4) E denotes a CA enzyme and B indicates the solvent buffer molecule (5). Histidine residue 64 of the CAII molecule functions as a proton shuttle in the rate-limited step for CO2 hydration at high substrate and buffer concentrations. Replacement of His-64 with non-ionizable amino acids such as alanine or glutamine markedly reduces hydratase activity.27.Tu C.K. Silverman D.N. Forsman C. et al.Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant.Biochemistry. 1989; 28: 7913-7918Crossref PubMed Scopus (338) Google Scholar,28.Engstrand C. Forsman C. Liang Z. Lindskog S. Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II.Biochim Biophys Acta. 1992; 1122: 321-326Crossref PubMed Scopus (22) Google Scholar Catalytic rates exhibited by CAII are among the highest measured (106 s−1)26.Silverman D.N. Lindskog S. The catalytic mechanism of carbonic anhydrases: implications of rate-limited protolysis of water.Acc Chem Res. 1988; 21: 30-36Crossref Scopus (670) Google Scholar (Figure 2). Reduced activity of some CA isoforms (CAIII), as well as species differences in CA isoform activity, provides additional insights into the function of other amino-acid residues within the CA catalytic domain. The cytosolic CAIII isoform that is abundantly expressed in skeletal muscle and adipose tissue and to a lesser extent in brain and liver, exhibits the lowest hydratase activity of the CA isoforms (see Figure 2). Although CAII and CAIII have similar backbone conformations (see Figure 1), CAIII is less efficient at proton shuttling due in part to replacement of His with lysine at residue 64.29.Jewell D.A. Tu C.K. Paranawithana S.R. et al.Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis.Biochemistry. 1991; 30: 1484-1490Crossref PubMed Scopus (83) Google Scholar In addition, residue 198 of CAIII contains a phenylalanine phenyl ring about 5 Angstrom from Zn whereas a leucine is present in CAII, perhaps resulting in steric constriction of the CAIII active site. Consistent with this supposition, replacement of Phe with leucine at position 198 increases the rate of CAIII catalysis by at least 10-fold.30.Duda D.M. Tu C. Fisher S.Z. et al.Human carbonic anhydrase III: structural and kinetic study of catalysis and proton transfer.Biochemistry. 2005; 44: 10046-10053Crossref PubMed Scopus (65) Google Scholar The amino-acid residues adjacent to His-64 also influence CAII activity.31.Jackman J.E. Merz Jr, K.M. Fierke C.A. Disruption of the active site solvent network in carbonic anhydrase II decreases the efficiency of proton transfer.Biochemistry. 1996; 35: 16421-16428Crossref PubMed Scopus (53) Google Scholar Introduction of bulky amino acids at position 65 decreases CAII Kcat up to 26-fold by disrupting the active site solvent network that facilitates proton transfer. In rodent CAIV (mouse, rat) a glutamine resides at position 63, and as a result the rodent enzyme has 10–20% of the activity of CAIV orthologs with a glycine at position 63 (rabbit, bovine, and human).32.Tamai S. Waheed A. Cody L.B. Sly W.S. Gly-63 → Gln substitution adjacent to His-64 in rodent carbonic anhydrase IVs largely explains their reduced activity.Proc Natl Acad Sci USA. 1996; 93: 13647-13652Crossref PubMed Scopus (21) Google Scholar The presence of a bulkier side chain at position 63 may alter the conformation of His64 and thereby decrease the efficiency of the proton shuttling reaction. The importance of CAIV in renal physiology is underscored by the fact that the 80% decrease in CAIV activity in rodents owing to the glutamine residue at position 63 is associated with the compensatory expression of additional CA isoforms in rodent kidney (e.g. CAXIV and CAXV; see below). The active site of CAII also contains a hydrophobic pocket, which includes valine at residues 121 and 143.33.Fierke C.A. Calderone T.L. Krebs J.F. Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II.Biochemistry. 1991; 30: 11054-11063Crossref PubMed Scopus (119) Google Scholar Substitution of Val143 with amino acids containing large side chains dramatically reduces hydratase activity. For example, a Val143 → Iso substitution reduces activity eightfold whereas Val143 → Tyr virtually destroys enzyme activity by reducing Kcat/Km for CO2 hydrations 3000–105-fold.33.Fierke C.A. Calderone T.L. Krebs J.F. Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II.Biochemistry. 1991; 30: 11054-11063Crossref PubMed Scopus (119) Google Scholar Blockade of the hydrophobic pocket with large amino-acid side chains may hinder the ability of CO2 to approach the zinc-hydroxide moiety with correct orientation to react. Overexpression of the CAII V143Y mutant displaces endogenous wild-type CAII from putative CAII binding sites and thereby functions as a dominant-negative mutant that may be used to assess the function of CAII interactions in facilitation of ion transport (see below). CAII is expressed as a 29 kDa monomeric protein (259 amino acids) in the cytosol of a wide variety of tissues (e.g. bone, brain, eye, stomach, intestine, liver, pancreas, kidney, red blood cells, salivary glands, and uterus),34.Ferrell R.E. Stroup S.K. Tanis R.J. Tashian R.E. Amino acid sequence of rabbit carbonic anhydrase II.Biochim Biophys Acta. 1978; 533: 1-11Crossref PubMed Scopus (20) Google Scholar, 35.Curtis P.J. Withers E. Demuth D. et al.The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II.Gene. 1983; 25: 325-332Crossref PubMed Scopus (39) Google Scholar, 36.Stolle C.A. McGowan M.H. Heim R.A. et al.Nucleotide sequence of a cDNA encoding rat brain carbonic anhydrase II and its deduced amino acid sequence.Gene. 1991; 109: 265-267Crossref PubMed Scopus (15) Google Scholar and is characterized by its high enzymatic activity and inhibition by exposure to sodium dodecyl sulfate (SDS), owing to the absence of stabilizing disulfide bridges.7.Brion L.P. Zavilowitz B.J. Suarez C. Schwartz G.J. Metabolic acidosis stimulates carbonic anhydrase activity in rabbit proximal tubule and medullary collecting duct.Am J Physiol. 1994; 266: F185-F195PubMed Google Scholar, 8.McKinley D.N. Whitney P.L. Particulate carbonic anhydrase in homogenates of human kidney.Biochim Biophys Acta. 1976; 445: 780-790Crossref PubMed Scopus (59) Google Scholar, 37.Whyte M.P. Carbonic anhydrase II deficiency.Clin Orthop. 1993; 294: 52-63PubMed Google Scholar As noted above, CAII expression in the nephron accounts for 95% of total CA activity in the kidney, and studies describing the localization of CAII messenger RNA (mRNA) and protein expression in specific nephron segments was recently reviewed in detail.38.Schwartz G.J. Physiology and molecular biology of renal carbonic anhydrase.J Nephrol. 2002; 15: S61-S74PubMed Google Scholar In summary and generalizing from a variety of animals, CAII is expressed in proximal tubule, thin descending limb, thick ascending limb, and intercalated cells of the cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (Figure 3). The function of CAII in renal H+/HCO3− transport is perhaps best understood by examining CAII interactions with specific transporters. CAII binding to the carboxyl terminus of human AE1 (band 3) was the first report of direct interaction between CAs and members of the bicarbonate transporter (BT) family.39.Vince J.W. Reithmeier R.A.F. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl−/HCO3− exchanger.J Biol Chem. 1998; 273: 28430-28437Crossref PubMed Scopus (197) Google Scholar Evidence for CAII interaction with AE1 included: (1) CAII binding to a glutathione S transferase (GST)-AE1 C-terminal peptide fusion in vitro; (2) blockade of AE1/CAII binding by an antibody to the C-terminal region of AE1; (3) lectin-induced co-clustering of AE1 and CAII; and (4) co-precipitation of CAII with AE1.39.Vince J.W. Reithmeier R.A.F. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl−/HCO3− exchanger.J Biol Chem. 1998; 273: 28430-28437Crossref PubMed Scopus (197) Google Scholar CAII has since been shown to interact with additional members of the SLC4A family of BTs including three of the anion exchangers (AE1-3).39.Vince J.W. Reithmeier R.A.F. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl−/HCO3− exchanger.J Biol Chem. 1998; 273: 28430-28437Crossref PubMed Scopus (197) Google Scholar,40.Vince J.W. Carlsson U. Reithmeier R.A.F. Localization of the Cl−/HCO3− anion exchanger binding site to the amino-terminal region of carbonic anhydrase II.Biochemistry. 2000; 39: 13344-13349Crossref PubMed Scopus (92) Google Scholar Association of CAII with anion exchangers facilitates bicarbonate transport as demonstrated by the acetazolamide-mediated reduction of chloride/bicarbonate exchange activity in human embryonic kidney (HEK)293 cells transfected with AE1 complementary DNA.41.Sterling D. Reithmeier R.A.F. Casey J.R. A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers.J Biol Chem. 2001; 276: 47886-47894Crossref PubMed Scopus (289) Google Scholar In addition, AE1 mutants, unable to bind CAII, exhibit reduced HCO3− transport. Finally, overexpression of a functionally inactive CAII mutant (V143Y) inhibits chloride/bicarbonate exchange by anion exchangers by displacing wild-type CAII from its binding site on AE1.41.Sterling D. Reithmeier R.A.F. Casey J.R. A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers.J Biol Chem. 2001; 276: 47886-47894Crossref PubMed Scopus (289) Google Scholar Similar results were obtained for AE2 and AE3. CAII physically associates with anion exchangers (AE1-3) via an acidic motif in the C-terminal region of the transporter.39.Vince J.W. Reithmeier R.A.F. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl−/HCO3− exchanger.J Biol Chem. 1998; 273: 28430-28437Crossref PubMed Scopus (197) Google Scholar Although the COOH tail of AE1 contains four putative motifs, CAII binding is mediated only by the D887ADD motif.42.Vince J.W. Reithmeier R.A. Identification of the carbonic anhydrase II binding site in the Cl(−)/HCO(3)(−) anion exchanger AE1.Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (155) Google Scholar Binding to AE2 requires the homologous sequence, D887ANE.42.Vince J.W. Reithmeier R.A. Identification of the carbonic anhydrase II binding site in the Cl(−)/HCO(3)(−) anion exchanger AE1.Biochemistry. 2000; 39: 5527-5533Crossref PubMed Scopus (155) Google Scholar The corresponding interaction motif located in CAII is comprised of a basic patch of amino acids comprised of histidine and lysine in the N-terminal portion of the enzyme.40.Vince J.W. Carlsson U. Reithmeier R.A.F. Localization of the Cl−/HCO3− anion exchanger binding site to the amino-terminal region of carbonic anhydrase II.Biochemistry. 2000; 39: 13344-13349Crossref PubMed Scopus (92) Google Scholar Most if not all BT family members contain a consensus CAII binding motif consisting of a hydrophobic residue followed by at least two acid residues within the next four amino acids. CAII binding to members of the BT family is pH dependent. In vitro binding studies have consistently demonstrated that CAII binding to AE's and NBC3 (see below) is highest at pH 5 and binding affinity is markedly reduced at pH values above 7.39.Vince J.W. Reithmeier R.A.F. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl−/HCO3− exchanger.J Biol Chem. 1998; 273: 28430-28437Crossref PubMed Scopus (197) Google Scholar The pH dependence of CAII binding most likely reflects increased protonation of basic residues (lysine and histidine) within the transporter binding site of CAII leading to increased electrostatic interactions with acidic residues of the anion transporter. The influence of pH on the interactions between CAII and BT raises the possibility that the pH of the subplasmalemma microenvironment may regulate the stoichiometry of CAII-transporter association in a dynamic manner. Sodium bicarbonate co-transporters include the kidney isoform, kNBC1 (NBC1a), an electrogenic sodium BT that is expressed on the basolateral membrane of proximal tubule cells.43.Gross E. Kurtz I. Structural determinants and significance of regulation of electrogenic Na+-HCO3− cotransporter stoichiometry.Am J Physiol. 2002; 283: F876-F887PubMed Google Scholar The HCO3−: Na+ stoichiometry is shifted from 3:1 to 2:1 mode in response to phosphorylation of Ser982 by protein kinase A, and the stoichiometry shift is accompanied by flux reversal from efflux in the 3:1 mode to influx in the 2:1 mode.43.Gross E. Kurtz I. Structural determinants and significance of regulation of electrogenic Na+-HCO3− cotransporter stoichiometry.Am J Physiol. 2002; 283: F876-F887PubMed Google Scholar An acidic cluster of amino acids (D986 NDD), that is homologous to the (D887ADD) CAII binding site in AE1, is required for the phosphorylation-induced stoichiometry shift. CAII binding to kNBC1 was first demonstrated by isotherm calorimetry studies in which CAII bound with high affinity (Kd=160±10 nM) to a single site on a kNBC1 carboxy-terminus peptide (amino acids 915–1035: kNBC1915–1035).44.Gross E. Pushkin A. Abuladze N. et al.Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp986, Asp988 and kNBC1-carbonic anhydrase II binding.J Physiol. 2002; 544: 679-685Crossref PubMed Scopus (77) Google Scholar In kNBC1 there are two clusters of acidic amino acids, L958DDV and D986NDD, which are involved in binding to CAII, and site-directed mutagenesis studies reveal that the first aspartate residue in each cluster is critical for CAII binding.45.Pushkin A. Abuladze N. Gross E. et al.Molecular mechanism of kNBC1-carbonic anhydrase II interaction in proximal tubule cells.J Physiol. 2004; 559: 55-65Crossref PubMed Scopus (66) Google Scholar Facilitation of kNBC1-mediated bicarbonate transport by CAII was demonstrated in mouse proximal convoluted cell line cell transfectants in which mutations of the two putative CAII binding sites in kNBC1 exhibited a positive correlation between CAII binding and flux inhibition by acetazola