Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells

Mono-, di-, and trisulfonic acids, including 4,4′-diacetamido stilbene-2,2′-disulfonic acid (DAS) and 2-(4′-amino phenyl)-6-methylbenzene thiazol-3′,7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluoro-benzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, 1974). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements. The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8–1.2 ± 10⁶/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.     

Enhancement of anion permeability in lecithin vesicles by hydrophobic proteins extracted from red blood cell membranes.

Triton X-100 extracts of membrane proteins from ghosts of normal and pronase treated cells enhance the anion permeability of lecithin vesicles. With proteins from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate), a specific inhibitor of anion transport, the anion permeability is not enhanced. On the basis that the Triton X-100 extracts are considerably enriched in a protein component of 95,000 molecular weight (or a 65,000 molecular weight segment in the case of pronase treated cells), and that DIDS is bound almost exclusively to the same proteins, it is suggested that the pronase resistant, 65,000 molecular weight segment of the 95,000 molecular weight protein is directly involved in anion transport.     

Membrane effects of nitrite-induced oxidation of human red blood cells.

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.     

Mechanism of anion transport in red blood cells: role of membrane proteins.

A number of anionic chemical probes that inhibit anion permeability of red blood cells are localized in a membrane protein of about 100,000 daltons, known as band 3. The inhibitory site has been explored using a series of disulfonic stilbene compounds. It apparently contains three positive charges, probably amino groups. Two probes, pyridoxal phosphate and N-(4-azido-2-nitropheyny)-2-amino ethyl sulfonate, are transported by the anion system but can be fixed in an irreversible bond under specified conditions (reduction with NaBH4 or exposure to light, respectively). Data obtained with these compounds indicate that the inhibitory site in band 3 is the transport site itself. Band 3 protein is exposed in part on the outside of the cell but it is also hydrophobically associated with membrane lipid. A model is proposed in which the band 3 protein acts as an anion permeation channel through the lipid bilayer. Near the outer aspect of the channel an anion binding site can undergo a local conformational change allowing a one-for-one anion exchange across a diffusion barrier.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Membrane stress increases cation permeability in red cells.

The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping.     

Membrane permeability equations and their solutions for red cells

Summary The mathematical equations for the transport of nonelectrolytes across cell membranes are critically examined and cast in forms suitable for solution which involve fewer approximations than has heretofore been commonly done. For the case of red cells, the equations are developed to include the effect of the variation in apparent nonosmotic water owing to the variation in hemoglobin concentration as the cell swells or shrinks. Two methods of solution of the equations are developed and studied and sample calculations are provided. It is shown that the solutions to the linearized equations commonly found in the literature are insufficiently accurate for some purposes and this inaccuracy is avoided by the methods given here. The importance of retaining the effects of variations in apparent nonosmotic water and in solute volume in the cell is demonstrated.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Osmotic water permeability of human red cells

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives

Summary The disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of the red blood cell, was found to be largely reversibly bound. Reversal of its specific inhibitory effect on anion permeability was attained by washing the cells with buffer containing albumin. The small fraction of covalently bonded SITS could be increased by prolonging the time of exposure of the cells or by multiple exposures. A series of other disulfonic stilbene derivatives was synthesized. All of them specifically inhibited anion permeability whether or not they are capable of forming covalent bonds. Their inhibitory effectiveness, however, varied over a 5,000-fold range, allowing certain conclusions to be made concerning the chemical architecture of the binding site. Certain of the compounds were almost entirely covalently bonded. One of them was labeled with¹²⁵I and used to determine to which membrane proteins the compound is bound. Over 90% was found in a protein band on acrylamide gels of 95,000 mol wt. The most effective compound against sulfate permeability was equally effective against chloride permeability, producing a maximum inhibition of over 95%. The residual anion fluxes respond differently to pH and temperature than do the fluxes of unmodified cells.This paper is based on work performed in part under contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project (Report No. UR 3490-199) and in part by the Medical Research Council of Canada, Grant No. MA-4665. Z. I. Cabantchik was the recipient of an International Atomic Energy Agency (Vienna) Fellowship.