Inhibition of cation cotransport by cholesterol enrichment of human red cell membranes.

1. Human red cells were enriched with cholesterol by incubation with lipid dispersions having a high cholesterol: phospholipid mol ratio and the kinetics of the furosemide-sensitive cotransport for Na+ and K+ were measured. 2. Influxes of both K+ and Na+ through this system were inhibited by 70 and 76% in cholesterol-rich cells (cholesterol: phospholipid mol ratio 1.80) and the Km of the furosemide-sensitive flux components for both K+ and Na+ decreased. 3. Effluxes of both K+ and Na+ are inhibited by furosemide and the magnitudes of these furosemide-sensitive components are markedly decreased in cholesterol-rich cells. 4. The inhibitory effect of cholesterol enrichment on this carrier-mediated transport of cations suggests that cholesterol may either alter the position of the carrier or retard its movement within a more viscous membrane micro-environment.     

Inhibition of human red cell Nak-ATPase by magnesium and potassium.

The inhibition of NaK-ATPase (EC 3.6.1.3) from human red cells by Mg²⁺ is markedly dependent on the relative concentrations of Na⁺ and K⁺. Inhibition increases with increasing K⁺ and decreases with increasing Na⁺. The inhibition appears to be a combined effect of Mg²⁺ and K⁺ at sites distinct from the sites at which these cations activate the enzyme. The kinetics of activation of the enzyme by Na⁺ with inhibitory levels of Mg²⁺ and K⁺ are biphasic, indicating both low and high affinity Na⁺ sites. At noninhibitory levels of Mg²⁺ and K⁺ only high affinity Na⁺ sites are seen. The results are inconsistent with any model in which Mg²⁺ and K⁺ compete with Na⁺ at a single site. A kinetic model is proposed to explain the mechanism of inhibition by Mg²⁺ and K⁺.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Pressure and temperature effects on human red cell cation transport

Summary The effects of hydrostatic pressure and temperature on the three components of K⁺ uptake in human red cells have been investigated, using ouabain and bumetanide to distinguish between the pump, passive diffusion and cotransport. The pressure sensitivity for passive diffusion has been shown to depend on the counter-ion present. The order of this effect, Cl^–>Br^–>NO ₃ ^– >I^–, is the same as for the ionic partial modal volumes and the Hofmeister series. We have analyzed our experimental results thermodynamically, and propose a model for the activated transition-state complex of the potassium ion which involves the loss of water molecules from the secondary hydration shell, cosphere II.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb⁺ (K⁺) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb⁺ (K⁺) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca²⁺ (Gardos effect). The electron donors require the presence of intracellular Ca²⁺ to be effective, but at levels that do not stimulate by themselves the fluxes of K⁺. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca²⁺ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca²⁺ more effective to elicit rapid K⁺ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Inhibition of Ca2+-dependent K+ channels by lead in one-step inside-out vesicles from human red cell membranes

Pb2+ modified the apparent threshold sensitivity to Ca2+ of individual K+ channels with a biphasic time-course. At first, the sensitivity to Ca2+ was lowered with the result of a decrease of the fraction of activated vesicles at a given Ca2+ concentration. Later, Pb2+ increased the sensitivity to Ca2+ and the fraction of activated vesicles. The increase of Pb2+ concentration increased the extent of the initial inhibition but decreased its duration. The inhibitory effect was not observed when the addition of Ca2+ preceded the addition of Pb2+. The presence of Mg2+ in the incubation medium was also required. In the absence of Mg2+, Pb2+ decreased the rate of uptake of 86Rb, but no decrease in the fraction of activated vesicles could be demonstrated.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

Pyruvate kinase-catalyzed ATP-formation in human red blood cell membranes

Previous studies on the linkage between enzymatically catalyzed ATP-generating reactions in the red blood cell membrane and the sodium and potassium transport in the control of overall glycolysis of human erythrocytes were controversial. In this study a significant amount of pyruvate kinase activity is shown to be localized within the membrane. Membrane fragments produce 20.5 mumol of ATP per 10(10) membranes per hour from phosphoenolpyruvate and ADP. The kinetics of the membrane-localized pyruvate kinase do not differ from those of the enzyme from hemolysates. The results clearly document the presence of the second ATP-generating enzyme of glycolysis, pyruvate kinase, in human red blood cell membranes. The main fraction of the enzyme is deeply hidden in the lipid layers of the membrane. It can be demasked by mechanical desintegration of membranes at high levels of activity. It is suggested that the amount of the membrane-localized fraction of pyruvate kinase is related to the clinical severity of the hemolytic process in pyruvate kinase deficiency.     

Non-selective voltage-activated cation channel in the human red blood cell membrane

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane.

When human red cells are incubated at 37 degrees C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, upcouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect. The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Effects of the plant alkaloid sanguinarine on cation transport by human red blood cells and lipid bilayer membranes

Summary The plant alkaloid, sanguinarine, inhibits the ouabain-sensitive K–Na pump and increases the downhill, ouabain-insensitive movements of K and Na in human red cells. These two effects have different temporal and concentration dependencies and are mediated by two different chemical forms of sanguinarine. The oxidized, charged form (5×10^–5 m) promptly inhibits the pump but does not affect leakage of K and Na. The reduced, uncharged form of sanguinarine causes lysis of red cells but does not inhibited the pump. Sanguinarine also increases the conductance of bilayers formed from sheep red cell lipids. The effect is produced by the uncharged but not by the charged form of sanguinarine. Bilayer conductance increases as the fourth power of sanguinarine concentration when the compound is present on both sides of the membrane and as the second power of concentration when present on only one side. Conductance also increasee-fold for each 34 mV increase in the potential difference imposed across the membrane. The results suggest that the uncharged forms of sanguinarine produce voltage-dependent channels in bilayers.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Influence of lead ions on cation permeability in human red cell ghosts

Summary Intracellular Pb²⁺ ions can replace Ca²⁺ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of⁸⁶Rb efflux by internal Pb²⁺ is around 5×10^–10 m, and stimulation is half-maximal at about 2×10^–9 m, and maximal at 10^–8 m Pb²⁺. There is no effect on²²Na efflux in this concentration range.⁸⁶Rb efflux is antagonized by internal Mg²⁺ ions, and by the channel-blocking drugs quinidine and diS-C₂(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb²⁺ ions, external Pb²⁺ increases both²²Na and⁸⁶Rb permeability when its concentration exceeds 6×10^–7 m. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb²⁺ ion buffers, and gives information about their preparation and properties.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.