Transport parameters in the human red cell membrane: solute-membrane interactions of amides and ureas.

We have studied the permeability of a series of hydrophilic amides and ureas through the red cell membrane by determining the three phenomenological coefficients which describe solute-membrane interaction: the hydraulic permeability (Lp), the phenomenological permeability coefficient (omega i) and the reflection coefficient (sigma i). In 55 experiments on nine solutes, we have determined that the reflection coefficient (after a small correction for solute permeation by membrane dissolution) is significantly less than 1.0 (P less than 0.003, t-test), which provides very strong evidence that solute and water fluxes are coupled as they cross the red cell membrane. It is proposed that the aqueous channel is a tripartite assembly, comprising H-bond exchange regions at both faces of the membrane, joined by a narrower sieve-specific region which crosses the lipid. The solutes bind to the H-bond exchange regions to exchange their solvation shell with the H-bonds of the channel; the existence of these regions is confirmed by the finding that the permeation of all the amides and ureas requires binding to well-characterized sites with Km values of 0.1-0.5 M. The sieve-specific regions provide the steric restraints which govern the passage of the solutes according to their size; their existence is shown by the findings that: (1) the reflection coefficient (actually the function [1-corrected sigma i]) is linearly dependent upon the solute molecular diameter; and (2) the permeability coefficient is linearly dependent upon solute molar volume. These several observations, taken together, provide strong arguments which lead to the conclusion that the amides and urea cross the red cell membrane in an aqueous pore.

Interrelation of ethylene glycol, urea and water transport in the red cell.

The reflection coefficient, sigma j, which measures the coupling between the jth solute and water transport across a semipermeable membrane, varies between 0 and 1.0. Values of sigma j significantly less than 1.0 provide irreversible thermodynamic proof that there is coupling between the transport of solute and solvent and thus that they share a common pathway. We have developed an improved method for measuring sigma and have used it to determine that sigma ethylene glycol = 0.71 +/- 0.03 and sigma urea = 0.65 +/- 0.03, in agreement with many, but not all, previous determinations. Since both of these values are significantly lower than 1.0, they show that there is a common ethylene glycol/water pathway and a common urea/water pathway. Addition of first one and then two methyl groups to urea increases sigma to 0.89 +/- 0.04 for methylurea and 0.98 +/- 0.4 for 1,3-dimethylurea, consistent with passage through an aqueous pore with a sharp cutoff in the 6-7 A region.

Sites of p-chloromercuribenzenesulfonate inhibition of red cell urea and water transport.

The mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate (pCMBS), inhibits water and urea fluxes across the human red blood cell membrane. The kinetics and affinities for pCMBS binding to separate water transport and urea transport inhibition sites were previously determined by Toon and Solomon ((1986) Biochim. Biophys. Acta 860, 361-375) in red cells that had been treated with N-ethyl-maleimide (NEM) to block five of the six sulfhydryls on the red cell anion exchange protein, band 3. We have used autoradiographs of gels from NEM-treated cells, labeled with 203Hg-pCMBS, to localize these water and urea transport inhibition binding sites separately and find that both are on band 3. Each site is saturable and the time course of each uptake can be fitted to the equation for a bimolecular association (with negligible dissociation) with time constants in agreement with those of Toon and Solomon. Determination of the binding stoichiometry shows one urea inhibition site and three water inhibition sites for every four band 3 molecules. These results indicate that band 3 plays a role in both urea and water transport and suggest that the functional unit may be a tetramer.

Control of red cell urea and water permeability by sulfhydryl reagents.

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.

Modulation of water transport in human red cells: effect of urea.

We have studied the effect of urea on water flux in the human red cell and have found that 500 mosmolal urea inhibits osmotic water transport by 39%. The Ki for urea inhibition of water flux is 550 +/- 80 mosmolal, higher than, but comparable with, the Km of urea transport into the red cell of 220-330 mM given by Mayrand and Levitt (J. Gen. Physiol. 55 (1983) 427) and Brahm (J. Gen. Physiol. 82 (1983) 1). Other amides, such as propionamide and valeramide, as well as methyl-substituted ureas, have similar effects, although an indifferent molecule, such as 0.5 M creatinine, has no effect. Urea can be washed off the inhibition site with buffer, and the effects of urea concentrations as high as 1.2 osmolal are entirely reversible. 500 mosmolal urea also significantly increases the reflection coefficient for ethylene glycol, sigma eth gly, from 0.71 +/- 0.03 in control experiments to 0.86 +/- 0.04 (P less than 0.0005, t-test), and propionamide has a similar effect on sigma eth gly. These results show that urea can modulate ethylene glycol transport through the red cell membrane, and are consistent with, but not proof of, the presence of a single class of aqueous channels through which both ethylene glycol and urea enter the red cell. It is suggested that the physiological purpose of these low-affinity urea sites is to modulate water flow out of the red cell during passage through the regions of 0.5-0.6 M urea in the kidney.

Effect of pCMBS on anion transport in human red cell membranes.

The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.

Relation between red cell membrane (Na+ + K+)-ATPase and band 3 protein.

This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na+ + K+)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na+ + K+)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170-9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the 31P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na+ + K+)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.

Inhibition of red cell urea flux by anion exchange inhibitors.

When they studied the chemical properties of red cell anion exchange inhibitors such as DIDS (4,4'-diisothiocyanate-2,2'-stilbene disulfonate), Barzilay et al. (1979) Membr. Biochem. 2, 227-254 also examined the benzene sulfonates. These molecules are structurally similar to half a DIDS molecule and are also specific anion exchange inhibitors with ID50 values measured in mM, rather than microM, as for the stilbene disulfonates. We have studied several inhibitors of the benzene sulfonate (BS) class and found that they also inhibit red cell urea flux by up to 92% and stimulate water flux by up to 58%. The values of Kinhib,app for urea flux inhibition are the same as the ID50 values for anion flux inhibition; covalent DIDS completely suppresses the inhibition. These observations strongly suggest that the effect on urea flux is caused by BS binding at the stilbene site. Comparative studies on the short chain amides exclude lipid solubility and solute molar volume as factors that affect these BS actions. Kstim,app for water flux stimulation is also related to the anion exchange ID50 values; covalent DIDS suppresses the water flux stimulation. These observations on urea and water fluxes are consistent with a common driver, located at the stilbene site, which is responsible for the BS actions on urea, water and anion fluxes. The subsequent steps are independent with separate effectors to modulate each of the individual fluxes. These effectors are presumably located in different regions of the protein or proteins and carry out their separate processes by allosteric means.

Effect of the sodium/potassium ratio on glyceraldehyde 3-phosphate dehydrogenase interaction with red cell vesicles.

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the 31P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K (1977) Biochim. Biophys. Acta 464, 82--92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation changed induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na4 + K+)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew (1970) J. Physiol. London 207, 393--402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by 31P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na+ + K+)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles.

We have characterized the asymmetric effect of Ca2+ on passive K+ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca2+, but not Mg2+, can induce an increase in K+ uptake in inside out vesicles. The half-maximal concentration of Ca2+ required to induce the K+ uptake is 0.2 mM, and the permeability increase is not specific for K+. Thus, the Ca2+- induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca2+ and is specific for K+. Removal of spectrin had no effect on the vesicle permeability increase due to Ca2+. Studies with N-ethylmaleimide show that the vesicle channel openings is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca2+-induced vesicle pathway is distinct from the normal channel for passive K+ leak in the absence of Ca2+. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca2+ -stimulated channel opening.

Permeability of human erythrocyte membrane vesicles to alkali cations.

The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using 86Rb as a K analog, we have measured the rate constant of 86Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.

Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles.

The temperature dependence of the binding of PhNapNH2 (N-phenyl-1-naphthylamine) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 degrees C, some 30 degrees C above the broad phase transition region of the phospholipid. In the temperature range above 20 degrees C, deltaH0 =--6100 cal-mol-1 and deltaS0 = 9.7 e.u.; in the temperature range below 20 degrees C, deltaH0 = 0 cal-mol-1 and deltaS0 = 30.4 e.u. These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 degrees C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 degrees C, though significantly greater than that above 20 degrees C, is still significiantly less than that in the crystalline state.

Role of membrane proteins and lipids in water diffusion across red cell membranes.

When human red cells are treated with the mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104-106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton T1 NMR and obtained a value of 4.0 X 10(-3) cm X s-1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of N-phenyl-1-naphthylamine by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation.

A systematic study has been made of the three coefficients that describe the human red cell membrane transport of a series of short straight-chain hydrophilic alcohols: the permeability coefficient, omega i, the reflection coefficient, sigma i, and the hydraulic conductivity, Lp. Ethylene glycol transport is saturable with Km = 220 +/- 50 mM; there is a second, low-affinity, ethylene glycol site which inhibits water transport (K = 570 +/- 140 mM, max. inhib. = 90 +/- 10%). sigma eth gly = 0.71 +/- 0.04 which is significantly less than 1 (n = 6, P less than 0.001), as are sigma i for six other alcohols (n = 23), thus providing strong thermodynamic evidence that water and these alcohols cross the red cell membrane, at least in part, in an aqueous channel. The solute/membrane frictional coefficient, fsm, for all seven alcohols has been determined and found to decrease monotonically as membrane permeability increases. The red cell membrane has been perturbed by treatments with phenylglyoxal and BS3 (bis(succinimidyl suberate]; these treatments are accompanied by correlated modulation of both ethylene glycol and urea permeability. In one set of experiments in control cells, urea permeability is correlated with water permeability; and, in another set, ethylene glycol permeability is correlated with water permeability. All of these observations support the proposition that the urea class of solutes, the ethylene glycol class of solutes and water all cross the membrane through the same aqueous pore. A schematic model of the red cell pore, consistent with the experimental observations, is presented.

Membrane mediated link between ion transport and metabolism in human red cells.

When 10(-6) M oubain is added to human red cell that have been incubated without glucose for two hours, there is a significant shift in the 31P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na+ + K+)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na+ + K+)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10(-6) M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na+ + K+)-ATPase and that the interaction is inhibited by 10(-6) M ouabain.

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape.

Na+ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na+ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 31P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg2+/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.

Ouabain-sensitive interaction between human red cell membrane and glycolytic enzyme complex in cytosol.

Binding of 2,3-diphosphoglycerate to monophosphoglycerate mutase, of which it is an obligatory cofactor, causes changes in the resonance positions of the 31P nuclear magnetic resonance spectra of both phosphate groups. It has previously been shown that these resonances shift when other glycolytic enzymes, such as phosphoglycerate kinase, are added to form the 2,3-diphosphoglycerate . monophosphoglycerate mutase . phosphoglycerate kinase complex. In view of this association, we have examined the set of glycolytic enzymes from aldolase to pyruvate kinase and found evidence of direct communication between all of these enzymes. A multi-enzyme complex of 1--2 . 10(6) daltons has been separated from broken cell ghosts by Biogel column filtration and evidence has been presented to show that this complex exhibits aldolase, glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase activity. The glycolytic multi-enzyme complex interacts with the outer face of inside-out vesicles prepared from human red cells and the interaction is suppressed by application of 10(-6) M ouabain to the inner face of these vesicles. These studies show that the conformation of the enzymes comprising the megadalton complex are responsive to the application of ouabain to the outer red cell membrane surface.

Relation between red cell anion exchange and urea transport.

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.

Urea reflection coefficient for the human red cell membrane.

The reflection coefficient, sigma, is an irreversible thermodynamic parameter which measures the interaction between solute and solvent in passage across a membrane. The initial estimate of Goldstein and Solomon ((1960) J. Gen. Physiol. 44, 1-17) by the zero-time method gave sigma urea = 0.6 for the human red cell membrane and a more recent measurement by Levitt and Mlekoday ((1983) J. Gen. Physiol. 81, 239-253) using a different method gave sigma urea = 0.95. We have now developed a variant of the zero-time method which gives sigma urea = 0.70 +/- 0.02, which is significantly different from 1.0. There has been controversy as to whether urea permeates the human red cell by the same channel used by water or by a different route. The finding that sigma urea is significantly less than 1.0 (actually less than 0.95) makes it possible to discriminate between these two possibilities since completely independent transfer of urea and water mandates a value of sigma urea = 0.95. Values significantly lower than 0.95 can only be achieved if the transport of the solute, urea, is coupled to that of the solvent, water.

Observations on Levitt's "new theory of transport".

Levitt (1974) (Biochim. Biophys. Acta 373, 115--131) has recently developed a "New Theory of Transport for Cell Membrane Pores" based on the supposition that equivalent pores in the red cell membrane are so small that water and small solute molecules such as urea can not pass each other. Levitt's concept is based on the implicit assumption that urea and water are spherical molecules. We have shown, using a scale model, that Levitt's supposition is not in agreement with the actual molecular shapes. Levitt has further asserted that there is a serious methodological error in measurements reported fifteen years ago by Goldstein and Solomon (1960) (J. Gen. Physiol. 44, 1--17). We have shown that the supposed "methodological error" lies in the fact that Levitt made his mathematical analysis of the appropriate equations under conditions significantly different from those employed by Goldstein and Solomon. A computer solution of the equations under the actual conditions used shows that Levitt's assertion is not justified.