Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

Mediated transport of anions in band 3-phospholipid vesicles

Band 3 protein, extracted from human erythrocyte membranes by Triton X-100, was recombined with egg lecithin/cholesterol mixtures to form small unilamellar vesicles at a yield of 15–20%. These systems exhibited sulfate fluxes which were inhibitable by stilbene disulfonates and other inhibitors. Maximal inhibition could only be obtained when inhibitors were present at both membrane surfaces. Inhibitor constants $\text{I}{}_{50}$ were higher than in the native membrane. Quantitatively, transport function was retained at least 60%, as related to the amount of protein involved. Sulfate transport in the recombinates resembled transport in the native membrane with respect to temperature dependence ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 29?32}\text{kcal}\text{/}\text{mol}$), pH dependence between pH 6.5 and 7.8, and the relationship between net and exchange fluxes. In contrast to the native cell, concentration dependence was linear up to 80 mM sulfate, which may be indicative of a lowered affinity for the substrate. Lactate transport in these systems, although substantial, was insensitive to stilbene disulfonates as well as to mercurials, indicating that band 3 is not involved in the specific monocarboxylate transfer in the erythrocyte. Anion transport in band 3-lipid recombinates was insensitive to cholesterol between 0 and 27 mol%. Treatment with proteases, while not affecting transport per se, abolished sensitivity to stilbene disulfonate inhibitors. These observations indicate a number of disturbances of band 3 after recombination, in spite of a preservation of the major transport properties.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

A relationship between anion transport and a structural transition of the human erythrocyte membrane

Scanning microcalorimetry was employed as an aid in examining some structural features of the anion transport system in red blood cell vesicles. Two structural transitions were previously shown to be sensitive to several covalent and non-covalent inhibitors of anion transport in red cells. In this study, these transitions were selectively removed, either thermally or enzymatically, and the subsequent effect on ³⁵SO₄^2? efflux in red cell vesicles was determined. It is shown that removal of one of these transitions (B₂) has a negligible inhibitory effect on anion transport. Cytoplasmic, intermolecular disulfide linkages between band 3 dimers are known to form during the B₂ transition. The integrity of the 4,4′-diisothiocyanostilbene-2,2′-disulfonate-sensitive C transition, on the other hand, is shown to be a requirement for anion transport. The localized region of the membrane giving rise to this transition contains the transmembrane segment of band 3, as well as membrane phospholipids. The calorimetric results suggest a structure of band 3 which involves independent structural domains, and are consistent with the transmembrane segment playing a direct role in the transport process.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.     

Kinetic analysis of l-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system

Three parallel pathways of l-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternately to the outside and the inside of the membrane.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Amino acid transport via the red cell anion transport system

Evidence is presented that the red cell anion-exchange transport (Band 3) can selectively transport small neutral amino acids, including glycine, serine and cysteine, but not alanine, proline, valine and threonine. This transport is inhibited by micromolar concentrations of SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate), and increased by raising the pH from 6.5 to 8.5.     

Formation of aqueous pores in the human erythrocyte membrane after oxidative cross-linking of spectrin by diamide

Oxidation of erythrocyte membrane SH-groups by diamide and tetrathionate induces cross-linking of spectrin (Haest, C.W.M., Kamp, D., Plasa, G. and Deuticke, B. (1977) Biochim. Biophys. Acta 469, 226–230). This cross-linking was now shown to go along with a concentration- and time-dependent enhancement of membrane permeability for hydrophilic nonelectrolytes and ions. The enhancement is specific for oxidative SH-group modifications, is reversible by reduction of the induced disulfides, can be suppressed by a very brief pre-treatment of the cells with low concentrations of $\text{N-}\text{ethylmaleimide}$ and is strongly temperature-dependent. The pathway of the induced permeability discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{3–8}\text{kcal/mol}$). These findings are reconcilable with the formation of a somewhat inhomogeneous population of aqueous pores with radii probably $\text{? 0.65}\text{nm}$. Estimated pore numbers vary with the size of the probe molecule. Assuming a diffusion coefficient as in bulk water within the pore, at least 20 pores per cell have to be postulated; more realistic lower diffusion coefficients increase that number. Alterations of the lipid domain by changes of cholesterol contents and insertion of hexanol or nonionic detergents alter the number or size of the pores. Since aggregation of skeletal and intrinsic membrane proteins also occurs after the SH-oxidation, in parallel to the formation of membrane leaks, one may consider (a) defects in the disturbed bilayer interface, (b) a mismatch between lipid and intrinsic proteins or (c) channels inbetween aggregated intrinsic proteins as structures forming the pores induced by diamide treatment.     

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein I. Covalent modification and inhibition of phosphate transport

The hydrophobic probe phenylisothiocyanate is utilized for chemical modification of human erythrocyte band 3 protein. The binding of phenylisothiocyanate to this protein is characterized in whole erythrocytes, erythrocyte ghost membranes and in isolated band 3 protein. The label, reactive with nucleophiles in their deprotonated form, is found in all three preparations to be covalently bound to band 3 protein. Under saturation conditions, 4–5 mol phenylisothiocyanate are covalently bound per mol protein (molecular weight 95 000). The described modification effects inhibition of phosphate entry into erythrocytes. 50% inhibition of phosphate transport is obtained following a preincubation of erythrocytes with 0.45 mM phenylisothiocyanate. Both phenylisothiocyanate binding and transport inhibition are saturating processes. The relationship of the two parameters is non-linear.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties

The transmembrane movements of lactate and other monocarboxylate anions in mammalian erythrocytes have been claimed, by virtue of their sensitivity to SH-reagents, to involve a transfer system different from the classical anion system (Deuticke, B., Rickert, I. and Beyer, E. (1978) Biochim. Biophys. Acta 507, 137–155). Inhibition of monocarboxylate transfer by SH-reagents, however, was incomplete to an extent varying for different monocarboxylates. The transport component insensitive to SH-reagents has now been shown to involve (a) the classical anion-exchange system, as demonstrated by sensitivity to specific disulfonate inhibitors, and (b) nonionic diffusion, as indicated by the characteristic pH- and concentration dependency of this component and its stimulation by aliphatic alcohols. Under physiological conditions about 90% of total lactate movement proceed via the specific system, 5% via the classical anion-transfer system, 5% by nonionic diffusion. These three components of lactate exchange differ in their activation energies. The specific lactate system mediates net fluxes almost as fast as exchange fluxes, in marked contrast to the classical anion-exchange system which mediates halide exchange much faster than halide net movements. The underlying mechanism, for maintenance of electroneutrality, is an OH^?-antiport or an H⁺-symport as indicated by the particular response of lactate net fluxes to changes of intra- or extracellular pH.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds II. Chemical properties of the flufenamate-binding site on the band 3 protein

Flufenamate is a powerful inhibitor of anion exchange in red blood cells. It binds to the band 3 protein involved in the transport as discussed in the preceding paper (Cousin, J.-L. and Motais, R. (1982) Biochim. Biophys. Acta 687, 147–155). The present study is concerned with the chemical properties of the inhibitory binding site. Structure-activity studies were performed with two sets of compounds derivated from anthranilate (considered as the basic structure of flufenamate). The molar concentrations required to produce 50% inhibition ($\text{I}{}_{50}$) varied over more than a 10⁴ range. The inhibitory activity was quantitatively correlated with the hydrophobic character of the molecules and the electron-withdrawing capacity of the substituents. Comparison between the inhibitory potency of flufenamate analogs made a definition of the contribution of each part of the molecule in the binding to the receptor possible. The results suggest that anionic inhibitors bind to a site which presents a positively charged groups at the water-protein interface whereas the hydrophobic part of the molecule is inserted into an hydrophobic and electron-donor region of the protein. The specificity of amphiphilic compounds towards anion transport is discussed.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+-and H+-gradients

In the presence of a Na⁺-gradient (out > in), l-glutamic acid and l-and d-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na⁺ by K⁺. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na⁺-dependent uphill transport of l-glutamic acid was stimulated by K⁺ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K⁺-gradient (in > out). Quantitative and qualitative differences in the K⁺ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in $\text{Na}{}^{\mathrm{+}}\text{-(out > in) + K}{}^{\mathrm{+}}\text{-(in > out)}$ gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na⁺-gradient and the Na⁺ + K⁺-gradient-dependent transport of l-glutamic acid. However, pH- or K⁺-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of l-glutamic acid transport in which Na⁺ and K⁺ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na⁺ in the protonated or unprotonated forms, respectively. We also propose that K⁺ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Oligomeric structure and the anion transport function of human erythrocyte band 3 protein

Conclusions Evidence from many laboratories using several different techniques strongly suggests that, in the intact red cell, band 3 exists as dimers which can associate with other dimers to form tetramers. The kinetics of anion transport inhibition by stilbenedisulfonates indicate that irreversible inhibition of one subunit does not detectably affect anion transport by the other subunit. This does not imply that monomeric band 3 could necessarily transport anions; the native conformation of each subunit may require stabilizing interactions with another subunit, as indicated by the recent work of Boodhoo and Reithmeier [10]. A more detailed understanding of the structure of the band 3 dimer/tetramer will require information on which specific segments of the primary structure are involved in subunit-subunit contact. The combination of chemical cross-linking with proteolysis [136] is a promising approach to this problem.