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.     

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.     

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.     

Proteolysis of band 3 is enhanced by anti-Rho(D) binding to the red cell membrane

Surface radioiodinated human red cells were incubated with IgG fractions and the radioelectrophoretic profile of the ghost membranes determined. The patterns of RhO(D)-negative membranes exposed to anti-RhO(D) IgG and RhO(D)-positive membranes exposed to non-immune IgG fractions remained intact. Membranes of RhO(D)-positive membranes following incubation with anti-RhO(D) IgG showed a sharp reduction in the quantity of intact band 3, the main glycoprotein of the red cell membrane. This process was significantly abrogated in the presence of protease inhibitors. The results suggest a possible role for IgG binding in promoting the generation of band 3-derived fragments described by others as normal constituents of isolated ghosts.     

The locations of the three cysteine residues in the primary structure of the intrinsic segments of band 3 protein,and implications concerning the arrangement of band 3 protein in the bilayer

The intrinsic domains of band 3 protein contain three cysteine residues, one in a 17 kDa middle segment and two in a 35 kDa C-terminal segment. The latter are retained in an 8 kDa fragment produced by chymotrypsin treatment of ghosts. Cleavage of cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) allows localization of this amino acid in the primary structure of the 8, 17, 35 and 52 (17 plus 35) kDa segments of band 3 protein. The mapping of these residues taken with other information concerning accessibility of various sites at the two sides of the membrane leads to the conclusion that band 3 protein crosses the membrane at least five times, or ten times in a dimer structure. The implications of this conclusion in terms of band 3 protein structure and function are briefly discussed.     

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.     

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.     

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     

Electron spin resonance studies on the inorganic-anion-transport system of the human red blood cell Binding of a disulfonatostilbene spin label (NDS-tempo) and inhibition of anion transport

The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily adsorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0–2.0 μM (pH 7.6, 20°C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5–2.5 μM (pH 7.3, 25°C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system.     

Trans to Cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane

Ionophore A23187-mediated net influx of Ca²⁺ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca²⁺-induced increase in K⁺ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K⁺ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca²⁺ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 μM extracellular Ca²⁺. With cellular pH fixed at various values, the net influx of Ca²⁺ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit ΔpH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca²⁺ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K⁺-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37ndash;44).     

Interactions of the chelating agent 2,3-dimercapto-propane-1-sulfonate with red blood cells in vitro. I. Evidence for carrier mediated transport

Uptake of the water soluble 1,2-dimercaptopropanol (BAL) derivative 2,3-dimercapto-1-sulfonate (DMPS) into human red blood cells was found in vitro and the mode of penetration studied in detail. The compound entered erythrocytes in a concentration dependent manner. In contrast to sealed ghosts where inside and outside concentrations reached the same value, DMPS accumulated in intact erythrocytes. Since no binding of DMPS could be detected, the reason for accumulation was assumed to be a conversion of DMPS into chelates or metabolites which penetrated the membrane in a slower rate. A facilitated transport of DMPS mediated by the anion carrier protein was concluded on the basis of the following similarities with the anion transport: inhibition of [¹⁴C]DMPS-uptake by N-ethylmaleimide (NEM), tetrathionate (90%), sulfate (50%), 5,5′-dithio bis(2-nitrobenzoic acid) (DTNB) (25%); inhibition of uptake and efflux by 4,4′-diisothiocyano-2,2′-stilbene disulfonate (DIDS) (80%), dipyridamole (55%); temperature dependency (activation energy 24 K_cal/mol); pH-dependency (pH optimum about 6.9); counter-transport; activation of uptake by preincubation with DMPS (transmembrane effect).