A. K. Solomon

The aqueous pore in the red cell membrane: band 3 as a channel for anions, cations, nonelectrolytes, and water.

This article develops arguments for the existence of an aqueous pore in the red cell membrane as the principal route for passive flux of ions, water, and small nonelectrolytes and proposes a molecular model for the pore. In principle, such an aqueous pore would provide easy passage into and out of the cell for all solutes small enough to enter the channel. The red cell membrane, however, regulates the fluxes of cations and anions closely and discriminates carefully among other small solutes. These constraints have been incorporated into the model, which visualizes the channel and its associated regulatory system as governing passive transport of ions of either sign, as well as water and small nonelectrolytes into and out of the cell. The model, which was formulated to consolidate a number of observations already in the literature, has caused us to look for new interrelations between inhibitors specific to cation, anion, and nonelectrolyte transport. The results of these experiments, presented below, demonstrate that interrelations d o exist and provide evidence that supports the view that a common aqueous channel provides primary access to the red cell cytoplasm.

Regulation of human red cell volume by linked cation fluxes.

SummaryOsmotic volume perturbations in human red blood cells lead to specific changes in cation fluxes. When the cells are shrunken, influx of both Na and K is increased and efflux of both cations is decreased. Thus, all four fluxes react to the stress in a cooperative sense to cause a net accumulation of cations so that water enters the cell to maintain osmotic equilibrium. Our data show that this process leads to a slow return to normal volume. The linkage observed between the several fluxes cannot be explained on the basis of a simple pump-leak hypothesis, but is consonant with a mechanism of volume regulation mediated by a conformational change in a membrane protein or protein complex.

Control of nonelectrolyte permeability in red cells

Abstract Ploretin is shown to alter the permeability coefficients, θ, of both hydrophilic and lipophilic solutes in human red cells membranes. At a phloretin concentration of 0.25 mM, the hydrophilic solute, urea, has an θ = 0.34 control whereas the lipophilic solute, 2,3-butanediol, has an θ = 2.1 control. Thus, phloretin, to which the membrane is impermeable, affects both hydrophilic and lipophilic permeation pathways. Our results are consistent with the view that phloretin interacts with a membrane protein to cause allosteric actions in which the conformational change effective in the aqueous path also controls lipid permeation.

Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3−/Cl− exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3−. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.

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.

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

SummaryInside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10−6 to 10−3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×105 and 6×103 M−1, and right side out vesicles exhibit three independent binding sites with association constants of 2×105, 1.4×104 and 3×102M−1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×105, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×105 per equivalent cell.

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.

The osmotic nature of the ion-induced swelling of rat-liver mitochondria

Abstract Analytical techniques have been developed to measure the relation between water uptake and cation uptake in rat-liver mitochondria. The water content of inner and outer mitochondrial compartments were separately measured using [ 131 I]albumin and [ 14C ]sucrose. The cation contents of the same mitochondrial pellets were measured using flame photometry. These data were used to measure the water content of each compartment which was necessary, since the basic osmotic relationships are only revealed when movements into the inner and outer compartments are separately examined. The relation between the water uptake and K + uptake, either spontaneous or valinomycin-induced, was dependent on medium osmolality. The concentration of the K + solution taken up in the inner compartment was compatible with the hypothesis that transport-linked mitochondrial swelling is driven by osmotic pressure differences which have been induced by solute movement. The changes in volume of the water compartment and in ion content during phosphate-induced swelling and during incubation in the absence of substrate were analyzed using the same methods. Under these experimental conditions also, the mitochondrial inner compartment appears to be in osmotic equilibrium with the external medium.

Effect of cell volume on potassium transport in human red cells

Abstract The influx of K + into fresh human red cells is shown to be reversibly dependent on cell volume. With a 10 % decrease in cell volume, K + influx increases by an average of 19 %. The effect persists in the presence of cardiac glycosides which are more effective inhibitors of K + influx in shrunken cells than in normal ones; furthermore, increases in extracellular K + inhibit the action of cardiac glycosides more in shrunken than in normal cells. It is suggested that the volume dependent flux change is due to a conformational change in a receptor on the cell surface. This hypothesis is consistent with observed changes in measurable characteristics of the transport mechanism.

Modulation of red cell K+ transport by membrane lipids

Alterations in the state of the membrane lipids affect human red cell K+ transport. Depletion of membrane cholesterol by 29–34% significantly inhibited both total K+ influx and ouabain-sensitive K+ influx. Addition of the hydrophobic anesthetic, chlorpromazine, in concentration from 2 · 10−5 to 2 · 10−4 M increased both total K+ influx and ouabain-sensitive K+ influx. In each case the effect on both processes was almost identical which indicates a linkage between K+ “pump” and “leak”. Further, these results demonstrate that red cell K+ transport can be modulated by local conditions in the micro-environment of the transport system.