Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl-/HCO3- exchanger, AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO2 to HCO3- + 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-/HCO3- exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl-/NO3- exchange assays, which were independent of CA activity, and in Cl-/HCO3- 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-/NO3- exchange activity with EC50 values in the range 0.22-2.8 microM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 microM of each compound was only 22-53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl-/HCO3- exchange assays, which depend on functional CA to produce transport substrate, 40 microM 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.     

An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1

Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ? HCO(3)(-) + 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(-)/HCO(3)(-) 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(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) 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(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.     

The substrate anion selectivity filter in the human erythrocyte Cl-/HCO3- exchange protein, AE1

AE1 facilitates Cl-/HCO3- exchange across the erythrocyte membrane. To identify residues involved in substrate selection and translocation, we prepared an array of single cysteine mutants in an otherwise cysteineless background. These mutants spanning the C-terminal portion of the AE1 membrane domain from Phe806-Cys885 were characterized for functional activity when expressed in human embryonic kidney 293 cells by measurement of changes of intracellular pH associated with bicarbonate transport. To identify residues involved in substrate translocation, transport activity was assessed for each mutant before and after treatment with the following sulfhydryl reagents: anionic para-chloromercuibenzenesulfonate; permeant (2-aminoethyl)methanethiosulfonate; and cationic [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Among the 80 mutants, only certain key residues in the Val849-Leu863 region were inhibited by the sulfhydryl reagent, consistent with direct involvement of these sites in anion transport. In the last two transmembrane segments, only mutants in the extracellular portion of the transmembrane segments could be inhibited by sulfhydryl reagent, suggesting that the outer portions line the translocation channel and the inner portions have some other role. Sensitivity to cationic MTSET and effects of Cl- identified the substrate charge filter as Ser852-Leu857.     

The bicarbonate transport metabolon

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 Cl- /HCO3(-) 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 HCO3(-) 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+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) 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 CAIV, 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."     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.     

Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)

The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl(-) for HCO(3)(-). However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), Asp(896)-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl(-)/HCO(3)(-) exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.     

Regulation of DRA and AE1 in rat colon by dietary Na depletion

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.     

Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line

A new transient expression system has been developed to investigate the function of anion exchangers in vivo. Human 293 cells were cotransfected with AE2 or AE3 cDNA together with a plasmid encoding a cell surface marker protein. Staining of the cells with antibody directed against a cell surface epitope present in the marker protein permitted the detection of cells expressing functional anion exchangers. Intracellular pH (pHi) recording in individual transfectants loaded with the fluorescent pHi indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, was used to determine the flux of HCO3- as a measure of Cl-/HCO3- exchange activity. Cells expressing either anion exchanger displayed significantly enhanced Cl-/HCO3- exchange activity compared with controls expressing only the marker. Transfection with either anion exchanger or with control plasmid resulted in altered intrinsic buffering capacity profiles compared with untransfected controls. Expression of either AE2 or AE3 did not result in changes in resting pHi. The activities of both AE2 and AE3 were stimulated at alkaline pHi, suggesting that an internal protonation site in AE2 and AE3 may regulate their activities. Both exchangers were inhibited reversibly and irreversibly by the anion 4,4'-diisothiocyanostilbene-2,2'-disulfonate with IC50 values of 142 and 0.43 microM for AE2 and AE3, respectively. These data indicate that structural differences in these highly conserved anion exchangers give rise to differences in affinities at the external anion binding site.     

AE anion exchangers in atrial tumor cells

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.     

Identification of the carbonic anhydrase II binding site in the Cl(-)/HCO(3)(-) anion exchanger AE1

The human Cl(-)/HCO(3)(-) anion exchanger (AE1) possesses a binding site within its 33 residue carboxyl-terminal region (Ct) for carbonic anhydrase II (CAII). The amino acid sequence comprising this CAII binding site was determined by peptide competition and by testing the ability of truncation and point mutants of the Ct sequence to bind CAII with a sensitive microtiter plate binding assay. A synthetic peptide consisting of the entire 33 residues of the Ct (residues 879-911) could compete with a GST fusion protein of the Ct (GST-Ct) for binding to immobilized CAII, while a peptide consisting of the last 16 residues (896-911) could not. A series of truncation mutants of the GST-Ct showed that the terminal 21 residues of AE1 were not required for binding CAII. Removal of four additional residues (887-890) from the Ct resulted in loss of CAII binding. Acidic residues in this region (D887ADD) were critical for binding since mutating this sequence in the GST-Ct to DAAA, AAAA, or NANN caused loss of CAII binding. A GST-Ct construct mutated to D887ANE, the homologous sequence in AE2, could bind CAII. AE2 is a widely expressed anion exchanger and has a homologous Ct region with 60% sequence identity to AE1. A GST fusion protein of the 33 residue Ct of AE2 could bind to CAII similarly to the Ct of AE1. Tethering of CAII to an acidic motif within the Ct of anion exchangers may be a general mechanism for promoting bicarbonate transport across cell membranes.     

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.     

Metabolon disruption: a mechanism that regulates bicarbonate transport

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3-. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl-/HCO3- exchanger with a suggested role in pancreatic HCO3- secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl-/HCO3- exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.     

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.     

Evidence for a second binding/transport site for chloride in erythrocyte anion transporter AE1 modified at glutamate 681

Transport kinetics have been examined in erythrocyte anion transporter AE1 that has been chemically modified to convert glutamate 681 to an alcohol (E681OH AE1). Outward conductive Cl(-) flux in E681OH AE1 is inhibited by removal of extracellular Cl(-); this effect is the opposite of that in native AE1 and is consistent with coupled electrogenic 2:1 Cl(-)/Cl(-) exchange. A second Cl(-) binding/transport site is also suggested by the characteristics of (35)SO(4)(2-) flux in E681OH AE1: bilateral and cis Cl(-), which are normally inhibitory, accelerate (35)SO(4)(2-) flux. These effects would be expected if Cl(-) binds to a second transport site on SO(4)(2-)-loaded E681OH AE1, thereby allowing Cl(-)/SO(4)(2-) cotransport. Alternatively, the data can be explained without proposing Cl(-)/SO(4)(2-) cotransport if the rate-limiting event for (35)SO(4)(2-)/SO(4)(2-) exchange is external SO(4)(2-) release, and the binding of external Cl(-) accelerates SO(4)(2-) release. With either interpretation, these data indicate that E681OH AE1 has a binding/transport site for Cl(-) that is distinct from the main transport site. The effects of graded modification of E681 or inhibition by H(2)DIDS are consistent with the idea that the new Cl(-) binding site is on the same E681OH-modified subunit of the AE1 dimer as the normal transport site.     

A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.     

Oxalate transport via the sulfate/HCO3 exchanger in rabbit renal basolateral membrane vesicles

We evaluated the mechanism of oxalate transport in basolateral membrane vesicles isolated from the rabbit renal cortex. An outward HCO3- gradient induced the transient uphill accumulation of oxalate and sulfate, indicating the presence of oxalate/HCO3- exchange and sulfate/HCO3- exchange. For oxalate, sulfate, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the K1/2 value for oxalate/HCO3- exchange was nearly identical to that for sulfate/HCO3- exchange, suggesting that both exchange processes occur via the same transport system. This was further supported by the finding of sulfate/oxalate exchange. Thiosulfate/sulfate exchange and thiosulfate/oxalate exchange were also demonstrated, but a variety of other tested anions including Cl-, p-aminohippurate, and lactate did not exchange for sulfate or oxalate. Na+ did not affect sulfate or oxalate transport, indicating that neither anion undergoes Na+ co-transport or Na+-dependent anion exchange in these membrane vesicles. Finally, we found that the stoichiometry of exchange is 1 sulfate or oxalate per 2 HCO3-, or a thermodynamically equivalent process. We conclude that oxalate, but not other organic or inorganic anions of physiologic importance, can share the sulfate/HCO3- exchanger in renal basolateral membrane vesicles. In series with luminal membrane oxalate/Cl- (formate) exchange, exchange of oxalate for HCO3- or sulfate across the basolateral membrane provides a possible transcellular route for oxalate transport in the proximal tubule.     

Kinetics of bicarbonate and chloride transport in human red cell membranes

Unidirectional [14C]HCO3- and 36Cl- efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0 degrees C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that 14CO2 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with CAn constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(eff) = J(eff)max.C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant Ki mM) to an intracellular site was included in the model (MS fit): J(eff) = J(eff)max.C(i)/[(K1/2 + C(i)).(1 + C(i)/Ki)]. The MM fits show that the external half-saturation constant, Ko1/2 ( = C(o)An for J(eff,o) = 1/2.j(eff,o)max) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM Ko1/2 is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(eff,o)max (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, Ki1/2 mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(eff,i)max (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO-3) and 0.50 (Cl-). The half-inhibition constants of the MS fit, Ki, are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, Ks1/2, is 20.2 (HCO-3) and 23.9 (Cl-) mM, and J(eff,s)max is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM Ks1/2 is 30.4 nM (HCO3-) and 50.1 mM (Cl-), and Ki is 541 mM (HCO3-) and 392 mM (Cl-).(ABSTRACT TRUNCATED AT 400 WORDS)     

Is CFTR an exchanger?: Regulation of HCO3−Transport and extracellular pH by CFTR

The disease, cystic fibrosis, is caused by the malfunction of the cystic fibrosis transmembrane conductance regulator. Expression of functional CFTR may normally regulate extracellular pH via control of bicarbonate efflux. Reports also suggest that the CFTR may be a Cl-/HCO3- exchanger. If true, this could be very important for treatment of CF given the airway host defense system is quite sensitive to pH, and acidic pH been found to increase mucus viscosity. We compared evidentiary support of four possible models of CFTR's role in the transport of bicarbonate: 1) CFTR as a Cl-channel that permits bicarbonate conductance, 2) CFTR as an anion Cl-/HCO3- exchanger (AE), 3.) CFTR as both a Cl-channel and an AE, and 4.) CFTR as a Cl-channel that allows for transport of bicarbonate and regulates an independent AE. The effect of stimulators and inhibitors of CFTR and AEs were evaluated via iodide efflux and studies of extracellular pH. This data, as well as that published by others, suggest that while CFTR may support and regulate bicarbonate flux it is unlikely it directly performs Cl-/HCO3- anion exchange.     

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.