Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

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.     

Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

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.     

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.     

Inhibition of anion transport in human red blood cells by 5,5′-dithiobis(2-nitrobenzoic acid)

The uptake of [³²P]phosphate into human red blood cells was inhibited ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.6}\text{mM}$) by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7}\text{mM}$). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[¹⁴C]-benzamido-4′-aminostilbene-2,2′-disulfonate, a potent inhibitor of anion transport ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1?2 μ}\text{M}$), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.     

Identification of the Cl− transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe

H₂DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl^? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H₂DIDS are consistent with the concept that Cl^? and H₂DIDS compete for the transport site of the anion exchange system. The value of K_i for H₂DIDS is 0.046 μM, indicating that H₂DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H₂DIDS occurs at the same site as the reversible binding, H₂DIDS can be used as a covalent label for the transport site. The specific localization of H₂DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.     

Site of red cell cation leak induced by mercurial sulfhydryl reagents

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS ($\text{p-}\text{chloromercuribenzene sulfonate}$) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that $\text{N-}\text{ethylmaleimide}$, a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.     

Interaction of thiourea with band 3 in human red cell membranes

Summary Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent,pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore,pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence ofpCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea,pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.     

Kinetic mechanism of chlorpromazine inhibition of erythrocyte 3-O-methylglucose transport

The kinetic mechanism of chlorpromazine inhibition of erythrocyte hexose transport was investigated using the non-metabolizable glucose analog $\text{3-O-}\text{methylglucose}$. It was found that chlorpromazine added to the external medium is a non-competitive inhibitor of both equilibrium exchange and net $\text{3-O-}\text{methylglucose}$ transport at pH 7.8, 15°C. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for equilibrium exchange is $\text{76 ± 21 μM}$. When net efflux and equilibrium exchange were measured on the same population of cells the equilibrium exchange was 2.5-times the maximum net efflux. The percent reduction of $\text{3-O-}\text{methylglucose}$ flux by chlorpromazine is dependent upon chlorpromazine concentration and not $\text{3-O-}\text{methylglucose}$ concentration as expected for a non-competitive inhibitor. Equilibrium exchange and net efflux show the same extent of inhibition at each concentration of chlorpromazine evaluated. These results suggest that exchange and net efflux of $\text{3-O-}\text{methylglucose}$ in the human erythrocyte may share a common transport system.     

Pyruvate flux into resealed ghosts from human erythrocytes

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ranged from 15.0 mM at $\text{μ = 0.05}$ to 3.7 mM at $\text{μ = 0.01}$, while the $\text{V}$ went from $\text{0.611 · 10}{}^{-15}$ to $\text{0.137 · 10}{}^{-15}\text{mol}\text{·}\text{min}{}^{-1}\text{·}\text{ghost}{}^{-1}$. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO₄ mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by stilbene derivatives and the identification of an inhibitor-binding protein

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H₂DIDS bound to the vesicles was determined by using [³H]-H₂DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [³H]H₂DIDS showed that about 10% of the total bound H₂DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H₂DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H₂DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.     

The quenching of an intramembrane fluorescent probe. A method to study the binding and permeation of phloretin through bilayers

Phloretin and phloretin-like dipolar non-electrolytes strongly quench the fluorescence of several membrane-bound probes, including 1,6-diphenylhexa-1,3,5-triene and anthroyl derivatives of long-chain fatty acids. Fluorescence intensity measurements therefore provide a simple and sensitive method to study the equilibrium binding properties and permeability of phloretin-like molecules in biological and artificial membrane systems. The dissociation constants for the binding of phloretin and naringenin to phosphatidylcholine vesicle membranes are determined, assuming the Stern-Volmer relation, from the fluorescence intensity of intramembrane probes as a function of phloretin and naringenin concentrations. Results (phloretin, $\text{9 ± 1}\text{μM}$; naringenin, $\text{21 ± 4}\text{μM}$) agree with the dissociation constants obtained using absorption titration performed in the absence of fluorescent probes. Fluorescence nanosecond lifetime measurements show that the mechanism of quenching of diphenylhexatriene and 16-anthroylpalmitic acid by phloretin and naringenin is largely diffusional in nature. The transmembrane movement of phloretin through phosphatidylcholine vesicles was observed by the stopped-flow technique, in which phloretin is mixed rapidly with a vesicle solution containing a membrane-bound fluorescent probe. The time course obtained by fluorescence measurements was identical to that obtained in a parallel measurement of the time course of optical absorption of phloretin. Stopped-flow data for the permeability of phosphatidylcholine liposomes and red blood cell membranes are also presented. The use of a membrane-bound indicator greatly extends the range of concentrations and pH values as well as the types of systems which can be characterized by optical means.     

Effects of 4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene on ion transport in turtle bladders

The disulfonic stilbene (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO₃^-−, Cl⁻) moeity of the short-circuiting current is eliminated by 4-acetamido-4′-isothiocyano-2,2′-disulfonic stibene, but only after its addition to the serosal bathing fluid. Whereas 4-acetmido-4′-isothiocyano-2,2′-disulfonic stilbene has no effect om Na⁺transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na⁺+K⁺)-ATPase of both turtle bladder and eel electric organ.     

Erythrocyte membrane potentials determined by hydrogen ion distribution

If the extracellular fluid is left unbuffered, dynamic membrane potential changes in the red blood cell may be determined from external pH readings. For some types of experiments it is necessary to accelerate H⁺ equilibration by adding minute amounts of hydrogen carriers. The method is independent of hematocrit over a wide range of membrane potential changes. Membrane potential jumps produced by permeability changes or by changes in ionic composition may be measured. The method provides a convenient means of measuring parameters of both the conductive and non-conductive anion pathways in the red cell.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Anion transport in red blood cells and arginine-specific reagents: The location of [14C]Phenylglyoxal binding sites in the anion transport protein in the membrane of human red cells

The reaction of phenylglyoxal, a reagent specific for arginine residues, with erythrocyte membrane at pH 7.4 results in complete inhibition of sulfate equilibrium exchange across human red cells. The inactivation was found to be concentration and time depenent. The binding sites of this reagent in the anion transport protein (band 3) under these conditions were determined by using [¹⁴C]phenylglyoxal. The rate of incorporation of the radioactivity into band 3 gave a good correlation with the rate of inactivation. Under conditions where the transport is completely inhibited about 6 mol [¹⁴C]phenylglyoxal are incorporated into 1 mol band 3. Treating the [¹⁴C]phenylglyoxalated ghosts at different degrees of inactivation with extracellular chymotrypsin showed that about two-thirds of these binding sites are located on the 60 kDa fragment.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

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.