- (2001) Volume 2, Issue 4
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Deborah Sterling1, Reinhart AF Reithmeier2, Joseph R Casey1*
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| Corresponding Author Joseph R Casey University of Alberta Department of Physiology Edmonton, AB Canada T6G2H7 Phone +1-780-492.7203 Fax +1-780-492.8915 E-mail joe.casey@ualberta.ca |
Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO2 + H2O HCO3 - + H+ . These enzymes therefore both produce HCO3 - for transport across membranes and consume HCO3 - that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO3 - across cells and epithelial layers. Plasma membrane anion exchange proteins (AE) transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO3 - transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII) via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl- /HCO3 - exchange activity in transfected HEK293 cells. Treatment of AE1- transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wildtype CAII from AE1, a loss of transport activity would be observed if binding to the AE1 Cterminus is required for transport.
Keywords |
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| Carbonate Dehydratase, Band 3 Protein, Ion Transport | ||||||
Abbreviations |
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| AE: anion exchanger; BCECFAM: 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester; CA: carbonic anhydrase; GST: glutathione-Stransferase | ||||||
| In any discussion of bicarbonate transport it is important to consider the role of the enzyme carbonic anhydrase. Carbonic anhydrase (CA) catalyzes the production of HCO3 - from CO2 and H2O and the consumption of HCO3 - by conversion [1]. Thus the enzyme supplies the HCO3 - substrate for transport and removes HCO3 - following transport. Modulation of carbonic anhydrase activity therefore provides a means to regulate the rate of HCO3 - transport. The family of mammalian carbonic anhydrases consists of at least 10 members with both cytosolic forms and forms with catalytic site anchored to the extracellular surface of the cell [1, 2]. In studies of HCO3 - transport physiology it is important to consider the location, expression level and regulation of CA as this can have profound effects on the rate of HCO3 - transport. | ||||||
| One important family of HCO3 - transporters are the sodium independent Cl-/HCO3 - exchangers, or anion exchangers (AE). The best known member of the family is erythrocyte AE1 (Band 3), but AE family members are very widely expressed across tissues, including isoforms AE2, AE3 [3, 4] and the very recently described AE4 [5]. AE proteins facilitate the one-for-one electroneutral exchange of Cl- for HCO3 - and thereby contribute to pH regulation, CO2 metabolism, volume regulation and maintenance of Cl- and HCO3 - levels. Each AE protein has two large and one small domain (Figure 1). The N-terminal cytoplasmic domains of 43-77 kDa interact with glycolytic enzymes and cytoskeletal elements [6]. The approximately 55 kDa membrane domains alone carry out the anion transport function [7]. The C-terminal region of approximately 33 amino acids extends from the membrane on the cytosolic surface [8, 9] (Figure 1). | ||||||
| This C-terminal cytosolic region can be considered a separate domain given recent evidence of its important functional role. The C-terminal tail of AE1 was shown to bind to CAII, the most catalytically active CA isoform of the erythrocyte [17]. Evidence for the interaction was: 1) lectin-induced clustering of AE1 induced clustering of CAII in erythrocytes membranes; 2) CAII co-immunoprecipitated with AE1; 3) an antibody directed against the C-terminal region of AE1 blocked the AE1/CAII interaction; 4) a glutathione-Stransferase (GST) AE1 C-terminus protein bound CAII [17]. Subsequent studies with GST fusion proteins of portions of the AE1 Cterminus indicated that the AE1/CAII interaction was ionic and mediated by the Nterminal most acidic sequence (DADD) of the AE1 C-terminus [16] (Figure 1). Mutation of the acidic residues of the DADD sequence resulted in loss of CAII binding [16]. Interaction between AE and CA may be common to all AE isoforms since similar acidic sequences are found in the C-termini of AE2, AE3 and AE4. However, a direct interaction with CAII has thus far been shown only for AE1 and AE2 [15]. A basic sequence at the CAII N-terminus was identified to interact with the DADD motif of AE1 [18]. Taken together this evidence lead to the proposal that the AE1/CAII complex forms a metabolon, a physical complex of enzymes in a linked metabolic pathway, for carbon dioxide metabolism [19]. What this proposal lacked is evidence for the physiological significance of the metabolon. Below is evidence that the interaction between CAII and AE1 is physiologically significant. | ||||||
| To examine the role of carbonic anhydrase in anion exchange activity, HEK293 cells were transfected with AE1 cDNA. Transport activity was measured in cells loaded with the pHsensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM). In each assay, cells were alternately perfused with Ringer’s buffer containing Cl-, or with Ringer’s buffer with Clreplaced by gluconate. All solutions were bubbled continuously with 5% CO2 in air. AE1- mediated efflux of HCO3 - acidifies the cell, while influx alkalinizes the cell (Figure 2). In Figure 2 intracellular pH increases going up the Y-axis. Cells were incubated with the membrane-permeant CA inhibitor, acetazolamide (100 μM), and the transport activity was measured. Acetazolamide does not inhibit anion exchange activity, as opposed to other CA inhibitors which inhibit anion exchange [20]. Comparison of the transport rates before and after treatment with acetazolamide indicated that the rate of HCO3 - efflux was inhibited by 85±1%, while HCO3 - influx was inhibited by 48±6%. The endogenous expression level of CA is not ratelimiting to anion exchange since overexpression of CAII 10-20 fold did not increase transport activity (not shown). These data clearly indicate that CA activity has a large role in maximizing the rate of AE1 activity. The asymmetry of the effect, that CA has a larger effect during HCO3 - efflux than during influx, is also interesting. This data shows that CA is important to HCO3 - transport because it produces the substrate for HCO3 - efflux. CA has a lower effect on the rate of HCO3 - influx. However, the two fold decrease in the rate of HCO3 - influx after treatment with acetazolamide indicates that CA enhances HCO3 - influx by conversion of HCO3 - to CO2, decreasing the intracellular HCO3 - concentration, increasing the size of the inwarddirected HCO3 - gradient, and thereby driving HCO3 - transport forward. | ||||||
| Although it is now clear that CAII interacts with the C-terminus of AE1, the next question is whether this interaction has physiological significance. CAII can alternately interact with AE1 or be found free in the cytosol (Figure 3). One possible role of the AE1/CAII interaction is to maximize the rate of bicarbonate transport, by limiting the distance that bicarbonate needs to diffuse to reach the transport site and to minimize local [HCO3 -] during influx (Figure 3). To test the significance of the AE1/CAII interaction we have preliminary results from two types of experiments. First we have measured the anion exchange rate of AE1 mutants of the acidic CAII binding site (Figure 1). The sequence DADD of the AE1 Cterminus was identified as responsible for the CAII/AE1 interaction. Two GST fusion proteins of the C-terminal region, with DADD mutated to NANN and AAAA, were previously constructed and shown not to bind CAII [16]. Using the anion transport assay described in Figure 2 we measured the anion exchange rate of wild-type AE1 and AE1 mutants containing the NANN and AAAA sequences in place of DADD. Each of these mutants had greatly reduced anion exchange activity (flux 2.9±0.6 and 4.0±1.1 mM H+ equivalents/min, respectively) relative to wild-type AE1 (40±0.6 mM H+ equivalents/min), yet both mutants were processed to the cell surface as well as wild-type AE1. The simplest explanation for the observation is that loss of the CAII binding site on the mutants results in a reduced transport rate. No role of the C-terminal region in anion transport mechanism has ever been shown. However, it is also possible that these mutants compromise the AE1 mechanism or protein folding, resulting in reduced transport activity. | ||||||
| To examine further the functional role of the CAII/AE1 interaction we performed a dominant negative CAII experiment (Figure 3). In this experiment HEK293 cells were transfected with AE1 + vector, AE1 + wild-type CAII and AE1 + CAIIV143Y. The V143Y mutant of CAII has catalytic activity that is approximately three thousand fold lower than wild-type CAII [21]. The rate of AE1 transport activity when expressed alone was 40±0.6 mM H+ equivalents/min and co-expressed with wildtype CAII was 38±1.4 mM H+ equivalents/min, which indicates that overexpresion of wild-type CAII had no significant effect on AE1 transport activity. In contrast, overexpresion of V143Y CAII reduced AE1 transport activity to 16±2.8 mM H+ equivalents/min, which represents a 60% reduction in transport activity. We interpret these results to mean that overexpression of CAII V143Y results in displacement of endogenous wild-type CAII from its binding site on the AE1 C-terminus. The resulting decrease in bicarbonate transport rate indicates that AE1 transport activity is most efficient when CAII is localized to the cytoplasmic surface of AE1. | ||||||
| Three lines of evidence indicate a significant role of carbonic anhydrase in the transport of bicarbonate by AE1. Treatment of cells with the CA inhibitor, acetazolamide, resulted in a major decrease in bicarbonate transport rate by AE1. Mutants of AE1 that lack the ability to bind CAII have decreased transport activity relative to wild-type AE1. Overexpression of a catalytically inactive form of CAII exerts a dominant negative effect upon the wild-type CAII and results in loss of anion exchange activity by wild-type AE1. We conclude that interaction of CAII with AE1 potentiates the rate of bicarbonate transport. | ||||||
| The significance of these findings to the study of bicarbonate transport and cystic fibrosis is three-fold. First, clearly it is important to consider the impact of CA in the cell physiology of bicarbonate transport processes. Which CA isoforms are expressed in the relevant tissues, where is it expressed and when? In studies of bicarbonate transporters expressed in heterologous expression systems, it is important to determine whether the physiologically relevant CA isoforms are present in the expression system cells. These findings are also important to the study of cystic fibrosis because they suggest that other bicarbonate transport proteins may interact directly with CA. Do other bicarbonate transporters also bind CA? What effect does this interaction have on transport rate? Finally, these findings suggest that one way to regulate bicarbonate transport is to regulate CA activity, by changes of CA expression, or modulation of the interaction between CA and the bicarbonate transporter. | ||||||
Acknowledgements |
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| Research in the laboratories of J.R.C. and R.A.F.R is funded by the Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation (J.R.C.). J.R.C. is a Scholar of the Alberta Heritage Foundation for Medical Research (AHFMR) and a New Investigator of CIHR. D.S. is supported by Graduate Studentships from AHFMR and the Heart and Stroke Foundation of Canada. | ||||||
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