Proton fluxes associated with erythrocyte membrane anion exchange

Summary Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942.Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloridesulfate exchange. The pH rise can be reversed by the addition of HCO ₃ ^– (4 m) or 2,4-dinitrophenol (90 m). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloríde exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972.In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Roth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

The effects of dansylation on the pH dependence of SO4(2-) and Cl- equilibrium exchange and on the H+/SO4(2-) cotransport across the red blood cell membrane

Treatment of the erythrocyte membrane with dansyl chloride leads to the following effects: (i) SO4(2-) transport is enhanced, Cl- transport is reduced. At maximal acceleration of sulfate exchange, Cl- exchange is only partially inhibited. The two effects are lineary related suggesting that the Cl- and SO4(2-) transporting forms of band 3 are derived from the same pool. (ii) The maximum of the pH dependence of SO4(2-) equilibrium exchange as measured at low sulfate concentrations is replaced by a plateau. It now resembles the pH dependence of Cl- exchange in untreated red cells. The pH dependence of SO4(2-) equilibrium exchange as measured at high sulfate concentrations is virtually unchanged after dansylation. The pH dependence of the partially inhibited Cl- equilibrium exchange across the dansylated membrane as measured at high chloride concentrations remains similar as in the untreated red cells but is somewhat less pronounced. (iii) SO4(2-)/H+ cotransport remains essentially unaltered after modification by dansyl chloride. The effects of dansylation are discussed in terms of a model similar to the titratable carrier model originally proposed by Gunn (Gunn, R.B. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Rorth, M. and Astrup, P., eds.), pp. 823-827, Munksgaard, Copenhagen).     

Plasma membrane Cl-/HCO3-exchangers: Structure,mechanism and physiology

Anion exchanger proteins facilitate the exchange of bicarbonate for chloride across the plasma membrane. When bicarbonate combines with a proton it undergoes conversion into CO2, either spontaneously, or catalyzed by carbonic anhydrase enzymes. The CO2/HCO3- equilibrium is the body’s central pH buffering system. Rapid bicarbonate transport across the plasma membrane is essential to maintain cellular and whole body pH, to dispose of metabolic waste CO2, and to control fluid movement in our bodies. Cl-/HCO3- exchangers are found in two distinct gene families: SLC4A and SLC26A. Differences in the tissue distribution, electrogenicity, and regulation of the specific anion exchanger proteins allow for precise regulation of bicarbonate transport throughout the human body. This review provides a look into the structural and functional features that make this family of proteins unique, as well as the physiological significance of the different anion exchangers.     

Intracellular pH regulation in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.     

Anion exchanger 1: Protean function and associations

Anion exchanger 1 (AE1) is the most abundant protein on the erythrocyte membrane and is also present on the basolateral surface of the alpha intercalated cell in the distal nephron. Mutations can cause either hereditary haemolytic red cell diseases, or hereditary distal renal tubular acidosis. Classically it mediates the electroneutral exchange of chloride for bicarbonate, as well as comprising an important mechanical component of the red cell membrane. It is increasingly recognised that it plays many other roles too: alternative anion transport, such as sulphate transport and proton and sulphate symport, associations with other erythrocyte membrane proteins as part of the AE1 macrocomplex, regulation of glycolysis and more recently cation transport through the so-called ‘leak’ pathway. These new functions and associations are reviewed in health and disease, and the role of AE1 as a putative regulator of cell volume is discussed.     

31P and 35Cl nuclear magnetic resonance measurements of anion transport in human erythrocytes

The exchange of anions across the erythrocyte membrane has been studied using 31P nuclear magnetic resonance (NMR) to monitor inorganic phosphate influx and 35Cl NMR to monitor chloride ion efflux. The 31P NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular pH to 6.4, while the intracellular pH was 7.3. The 35Cl NMR resonance for intracellular Cl- was so broad as to be virtually undetectable (line width greater than 200 Hz), while that of extracellular Cl-is relatively narrow (line width of about 30 Hz). The transports of Pi and Cl-were both totally inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a potent inhibitor of the band 3 protein. Since the 31P resonance of Pi varies with pH, intra- and extracellular pH changes could also be determined during anion transport. The extracellular pH rose and intracellular pH fell during anion transport, consistent with the protonated monoanionic H2PO4-form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPO24-form. The rates of Cl-efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The Cl-efflux was found to coincide with the influx of the monoanionic H2PO4-form of Pi.     

Chloride--bicarbonate exchange in red blood cells: physiology of transport and chemical modification of binding sites

About 80% of the CO2 formed by metabolism is transported from tissues to lungs as bicarbonate ions in the water phases of red cells and plasma. The catalysed hydration of CO2 to bicarbonate takes place in the erythrocytes but most of the bicarbonate thus formed must be exchanged with extracellular chloride to make full use of the carbon dioxide transporting capacity of the blood. The anion transport capacity of the red cell membrane is among the largest ionic transport capacities of any biological membrane. Exchange diffusion of chloride and bicarbonate is nevertheless a rate-limiting step for the transfer of CO2 from tissues to lungs. Measurements of chloride and bicarbonate self-exchange form the basis for calculations that demonstrate that the ionic exchange processes cannot run to complete equilibration at capillary transit times less than 0.5 s. The anion exchange diffusion is mediated by a large transmembrane protein constituting almost 30% of the total membrane protein. The kinetics of exchange diffusion must depend on conformational changes of the protein molecule, associated with the binding and subsequent translocation of the transported anion. We have characterized the nature of anion-binding sites facing the extracellular medium by acid-base titration of the transport function and modification of the transport protein in situ with group-specific amino acid reagents. Anion binding and translocation depend on the integrity and the degree of protonation of two sets of exofacial groups with apparent pK values of 12 and 5, respectively. From the chemical reactivities towards amino acid reagents it appears that the groups whose pK = 12 are guanidino groups of arginyl residues, while the groups whose pK = 5 are likely to be carboxylates of glutamic or aspartic acid. Our studies suggest that the characteristics of anion recognition sites in water-soluble proteins and in the integral transport proteins are closely related.     

Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.     

Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane

Intestinal epithelial membrane transport of L-lactic acid was characterized using rabbit jejunal brush-border membrane vesicles (BBMVs). The uptake of L-[(14)C]lactic acid by BBMVs showed an overshoot phenomenon in the presence of outward-directed bicarbonate and/or inward-directed proton gradients. Kinetic analysis of L-[(14)C]lactic acid uptake revealed the involvement of two saturable processes in the presence of both proton and bicarbonate gradients. An arginyl residue-modifying agent, phenylglyoxal, inhibited L-[(14)C]lactic acid transport by the proton cotransporter, but not by the anion antiporter. The initial uptakes of L-[(14)C]lactic acid which are driven by bicarbonate ion and proton gradients were inhibited commonly by monocarboxylic acids and selectively by anion exchange inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid and protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone, respectively. These observations demonstrate that L-lactic acid is transported across the intestinal brush-border membrane by multiple mechanisms, including an anion antiporter and a previously known proton cotransporter.     

pH homeostasis in promyelocytic leukemic HL60 cells

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.     

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."     

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.     

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange

Mouse embryos at the two-cell stage are able to recover from an alkaline load. We found that this recovery is mediated by sodium-independent bicarbonate/chloride exchange: intracellular pH (pHi) recovery from alkaline load is inhibited by the anion exchange inhibitor 4,4'-diisothiocyanostilbene disulfonic acid, lack of bicarbonate, or lack of chloride. The dependence of the pHi recovery on extracellular chloride concentration exhibits Michaelis-Menten kinetics. Furthermore, uptake of chloride is inhibited in a dose-dependent manner by extracellular bicarbonate. The Km for external chloride was found to be about 3 mM, with a Ki for external bicarbonate of about 2 mM. The exchanger is active above approximately pH 7.15. These results demonstrate that mouse embryos at the two-cell stage possess a sodium-independent bicarbonate/chloride exchange mechanism that is similar to that found in other mammalian cells. This bicarbonate/chloride exchanger appears to be the sole pHi-regulatory mechanism in the two-cell stage mouse embryo, since our previous results have shown that there are apparently no specific mechanisms active in these cells for relieving acid loads.     

Evidence from electron paramagnetic resonance for function-related conformation changes in the anion-transport protein of human erythrocytes

The erythrocyte membrane protein involved in anion transport (band 3) was isolated in its native lipid milieu in the form of leaky vesicles and then was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-meleimide (MalMe4PipO). The resulting electron paramagnetic resonance spectrum of band-3-bound MalMe4PipO was resolved into a rapid tumbling component and another, relatively immobile component. The percentage of the signal contributed by the mobile component (Q), was sensitive to various characteristic factors known to affect erythrocyte anion transport: Q was a hyperbolic function of chloride concentration displaying a half-saturation constant K1/2 similar to that of chloride transport. On the other hand Q showed a biphasic response to sulfate concentration, in line with the relatively high affinity of sulfate for the anion modifier site. Q was a saturable function of pH, either in presence of Cl- or SO4(-2), showing a pKa between pH 6.0 and 6.5, in analogy with the pH titration curve of Cl- and SO4(-2), transport. Spin-labelled vesicles treated with a covalent inhibitor of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, were markedly less susceptible to changes in Cl- concentration. It is suggested that the electron paramagnetic resonance spectrum of MalMe4PipO covalently bound to the band-3 protein, reports conformational changes which are related to the anion-transport function of this protein.     

Effects of bicarbonate on lithium transport in human red cells

Lithium influx into human erythrocytes increased 12-fold, when chloride was replaced with bicarbonate in a 150 mM lithium medium (38 degrees C. pH 7.4). The increase was linearly related to both lithium- and bicarbonate concentration, and was completely eliminated by the amino reagent 4, 4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). DIDS binds to an integral membrane protein (mol wt approximately 10(5) dalton) involved in anion exchange. Inhibition of both anion exchange and of bicarbonate-stimulated lithium influx was linearly related to DIDS binding. 1.1 X 10(6) DIDS molecules per cell caused complete inhibition of both processes. Both Cl- and Li+ can apparently be transported by the anion transport mechanism. The results support our previous proposal that bicarbonate-induced lithium permeability is due to transport of lithium-carbonate ion pairs (LiCO-3). DIDS-sensitive lithium influx had a high activation energy (24 kcal/mol), compatible with transport by the anion exchange mechanism. We have examined how variations of passive lithium permeability, induced by bicarbonate, affect the sodium-driven lithium counter-transport in human erythrocytes. The ability of the counter-transport system to establish a lithium gradient across the membrane decrease linearly with bicarbonate concentration in the medium. The counter-transport system was unaffected by DIDS treatement. At a plasma bicarbonate concentration of 24 mM, two-thirds of the lithium influx is mediated by the bicarbonate-stimulated pathway, and the fraction will increase significantly in metabolic alkalosis.     

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Fr?lich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.     

Mini Review

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 [Formula: See Text] 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 [Formula: See Text] 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 [Formula: See Text] co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) [Formula: See Text] 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."     

Modification of a carboxyl group that appears to cross the permeability barrier in the red blood cell anion transporter

A recently developed method for converting protein carboxyl groups to alcohols has been used to examine the functional role of carboxyl groups in the red blood cell inorganic anion-transport protein (band 3). A major goal of the work was to investigate the carboxyl group that is protonated during the proton-sulfate cotransport that takes place during net chloride-sulfate exchange. Three kinds of evidence indicate that the chemical modification (Woodward's reagent K followed by borohydride) converts this carboxyl to an alcohol. First, monovalent anion exchange is inhibited irreversibly. Second, the modification stimulates sulfate influx into chloride-loaded cells and nearly eliminates the extracellular pH dependence of the sulfate influx. (The stimulated sulfate influx in the modified cells is inhibitable by stilbenedisulfonate.) Third, the proton influx normally associated with chloride-sulfate exchange is inhibited by the modification. These results would all be expected if the titratable carboxyl group were converted into the untitratable, neutral alcohol. In addition to altering the extracellular pH dependence of sulfate influx, the chemical modification removes the intracellular pH dependence of sulfate efflux. The modification is performed under conditions in which the reagent does not cross the permeability barrier. The large effect on the intracellular pH dependence of sulfate transport suggests that a single carboxyl group can at different times be in contact with the aqueous medium on each side of the permeability barrier.