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Abstract Background Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. Methods We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. Results Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. Conclusions These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.

Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solidphase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity. [ABSTRACT FROM AUTHOR]

Anion exchanger 1 (AE1) is the plasma membrane Cl-/HCO3- exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H2O + CO2 ↔ HCO3- + H+. The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl-/HCO3- exchange activity to AE1 alone, as assessed by the flux of H+ equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl- concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H+ equivalents and Cl- of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl-/HCO3- exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO3- transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl-/HCO3- exchange activity, consistent with the HCO3- transport metabolon model. [ABSTRACT FROM AUTHOR]

Bicarbonate transport and metabolism are key elements of normal cellular function. Two alternate transcripts of anion exchanger 3 (AE3), full-length (AE3fl) and cardiac (AE3c), are expressed in central nervous system (CNS), where AE3 catalyzes electroneutral Cl-/HCO3- exchange across the plasma membrane of neuronal and glial cells of CNS. Anion exchanger isoforms, AE3fl and AE3c, associate with the carbonic anhydrases (CA) CAll and CAIV, forming a HCO3- transport metaboIon, to maximize HCO3- flux across the plasma membrane. CAXIV, with catalytic domain anchored to the extracellular surface, is also expressed in CNS. Here physical association of AE3 and CAXIV was examined by coimmunoprecipitation experiments, using mouse brain and retinal lysates. CAXIV immunoprecipitated with anti-AE3 antibody, and both AE3 isoforms were immunoprecipitated using antiCAXIV antibody, indicating CAXIV and AE3 interaction in the CNS. Confocal images revealed colocalization of CAXIV and AE3 in MUller and horizontal cells, in the mouse retina. Cl-/HCO3- exchange activity of AE3fl was investigated in transiently transfected human embryonic kidney 293 cells, using intracellular fluorescence measurements of BCECF, to monitor intracellular pH. CAXIV increased the rate of AE3fl-mediated HCO3- transport by up to 120%, which was suppressed by the CA inhibitor acetazolamide. Association of AE3 and CAXIV may represent a mechanism to enhance disposal of waste CO2 and to balance pH in excitable tissues. [ABSTRACT FROM AUTHOR]

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3- conversions and coupled HCO3- transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl-/HCO3- exchanger AE2 or the putative Cl-/HCO3- exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl-l/HCO3- exchangers to form a bicarbonate transport metaboIon. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO 2 to HCO 3 - +H + . Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl - /HCO 3 - exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl - /NO 3 - exchange assays, which were independent of CA activity, and in Cl - /HCO 3 - exchange assays. Transport was measured by following changes of intracellular [Cl - ] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl - /NO 3 - exchange activity with EC 50 values in the range 0.22-2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl - /HCO 3 - exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations. [ABSTRACT FROM AUTHOR]

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane C1-/HCO-3 anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 CO-3 transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO-3 co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) CI-/HCO-3 exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAW, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon." [ABSTRACT FROM AUTHOR]

Human NBC3 is an electroneutral Na+/HCO3- cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3- metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O ↔ HCO3- + H+ in many tissues. We investigated whether NBC3, like some Cl-/HCO3- exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with Kd = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAll binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 Dl135N/Dl136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the phi recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [γ-32P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3- transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. [ABSTRACT FROM AUTHOR]

Examines the functional and physical relations between deregulated in adenoma bicarbonate transporter and carbonic anhydrase II (CAII). Interaction between cytosolic CAII Minimization of diffusional distance between CAII and bicarbonate transporter; Mode of regulation; Assessment on the effects of CA inhibitors acetazolamide.

Bicarbonate is not freely permeable to membranes. Yet, bicarbonate must be moved across membranes, as part of CO[sub 2] metabolism and to regulate cell pH. Mammalian cells ubiquitously express bicarbonate transport proteins to facilitate the transmembrane bicarbonate flux. These bicarbonate transporters, which function by different transport mechanisms, together catalyse transmembrane bicarbonate movement. Recent advances have allowed the identification of several new bicarbonate transporter genes. Bicarbonate transporters cluster into two separate families: (i) the anion exachanger (AE) family of Cl[sup –] /HCO exchangers is related in sequence to the NBC family of Na[sup +] /HCO cotransporters and the Na[sup +] -dependent Cl[sup –] /HCO exchangers and (ii) some members of the SLC26a family of sulfate transporters will also transport bicarbonate but are not related in sequence to the AE/NBC family of transporters. This review summarizes our understanding of the mammalian bicarbonate transporter superfamily.Key words: bicarbonate transport, anion exchange, pH regulation, sodium/bicarbonate co-transport, chloride/bicarborate exchange, carbonic anhydrase.Le bicarbonate ne diffuse pas librement au travers des membranes. Cependant, le bicarbonate doit passer au travers des membranes, car il participe au métabolisme du CO[sub 2] et il sert à régler le pH cellulaire. Toutes les cellules de mammifères expriment des protéines de transport du bicarbonate afin de faciliter le flux transmembranaire du bicarbonate. Ces transporteurs du bicarbonate agissent par différents mécanismes de transport et ils catalysent ensemble le mouvement transmembranaire du bicarbonate. Des développements récents ont permis d'identifier plusieurs nouveaux gènes de transporteurs du bicarbonate. Les transporteurs du bicarbonate sont regroupés en deux familles. D'une part, il y a la famille AE des antiporteurs Cl[sup –] /HCO ayant une séquence apparentée à celle des cotransporteurs Na[sup +] /HCOHCO et des antiporteurs Cl[sup –] /HCO dépendants du Na[sup +] de la famille NBC. D'autre part, des transporteurs de sulfate de la famille SLC26a transportent également le bicarbonate, mais leur séquence n'est pas apparentée à la séquence des transporteurs de la famille AE/NBC. Cette revue résume nos connaissances actuelles de la superfamille des transporteurs du bicarbonate chez les mammifères.Mots clés : transport du bicarbonate, échange d'anions, régulation du pH, cotransport du sodium/bicarbonate, échange du chlorure/bicarbonate, anhydrase carbonique.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

The Bicarbonate Transport Meeting was held as a satellite meeting of the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology (CSBMCB): Membrane Proteins in Health and Disease. The meeting covered the modern history of bicarbonate transporter proteins and brought together the major workers in the field. Ron Kopito recounted the story of the first determination of the amino acid sequence for a bicarbonate transporter, AE1/Band 3, 25 years earlier while working with Harvey Lodish at Harvard, while Tomohiro Yamaguchi and Teruhisa Hirai presented up-to-date data on AE1 structure obtained using electron crystallography. The meeting further spanned the spectrum of bicarbonate transporters, with sessions devoted to Cl-/HCO3- exchangers, Na+/HCO3- co-transporters, the link to carbonic anhydrase, and the SLC26 family of bicarbonate transporters expressed broadly in humans, yeast, and bacteria. Le symposium sur le transport du bicarbonate s'est tenu en tant que réunion satellite du 53ième Congrès annuel de la Société canadienne de biochimie et de biologie moléculaire et cellulaire (SCBBMC), Protéines membranaires et pathologies associées. Le symposium a couvert l'histoire moderne des transporteurs du bicarbonate et a réuni les principaux acteurs du domaine. Ron Kopito a relaté l'historique de la première détermination de la séquence d'acides aminés du transporteur de bicarbonate AE1/Bande 3, 25 ans plus tôt, alors qu'il travaillait avec Harvey Lodish à Harvard, tandis que Tomohiro Yamaguchi et Teruhisa Hirai ont présenté les données les plus récentes sur la structure de AE1 obtenues par cristallographie électronique. Le symposium a couvert de manière plus approfondie le spectre des transporteurs du bicarbonate, présentant des sessions consacrées aux échangeurs Cl-/HCO3-, aux co-transporteurs Na+/HCO3-, au lien à l'anhydrase carbonique, ainsi qu'à la famille SLC26 des transporteurs du bicarbonate exprimés largement chez l'humain, la levure et la bactérie. [ABSTRACT FROM AUTHOR]

Monocarboxylate transporters(MCTs)mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation ofMCTtransport activity requires direct binding ofCAIVto the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. InhumanCD147, binding ofCAIVwas mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 inCAIVcan either act asHdonor orH acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase inMCTtransport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na+/H+ exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na+/H+ exchanger. CAII did not bind to acidic sequences in NHE 1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na+/H+ exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail. [ABSTRACT FROM AUTHOR]

Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pHi) and HCO[SUB3][SUP-] metabolism. Electrogenic NBC 1 catalyzes HCO[SUB3][SUB-] fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydrationdehydration of CO[SUB2]/HCO[SUB3][SUP-]. The physical and functional interactions of CAlV and NBC1 were investigated. NBC1 activity was measured by changes of phi in NBCl-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of phi recovery by 44 4- 3%, as compared to NBCl-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAlV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAlV interaction. NBCl-mediated phi recovery rate after acid load was inhibited by 40 + 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAll competes with endogenous CAll for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full phi recovery activity. We conclude that CAlV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAll and CAIV close to the NBC1 HCO[SUB3][SUP-] transport site maximizes the transmembrane HCO[SUB3,SUP-] gradient local to NBC1 and thereby activates the transport rate. [ABSTRACT FROM AUTHOR]