Interactions of lithium and protons with the sodium-proton exchanger of dog red blood cells

Passive movements of Li in dog red blood cells (RBC) ar like those of Na and protons in being stimulated by osmotic cell shrinkage and inhibited by amiloride. Li and protons have similar asymmetrical effects on Na-H exchange. When the intracellular fluid is made rich in Li or protons, Na-H exchange is stimulated. When the extracellular fluid is enriched in Li or protons, Na-H exchange is inhibited. In the case of protons, these effects can override alterations in driving force that are created by the experimental conditions. For example, acidification of the cytoplasm stimulates outward Na movements, while acidification of the medium inhibits Na efflux. Thus, protons (and, by analogy, Li) can interact with the Na-H exchanger not only as substrates but also as modulators. In previous experiments, the only way to activate the Na-H exchanger in dog RBC was to shrink the cells in hypertonic media. The influences of Li or protons, however, are so strong as to preempt the volume effects, so that the pathway can be activated even in swollen cells and deactivated in shrunken ones.     

Kinetics and regulation of the ankyrin-band 3 interaction of the human red blood cell membrane

In an attempt to identify potential regulatory mechanisms for erythrocyte membrane-cytoskeletal interactions, the kinetics and pH dependence of the band 3-ankyrin interaction were investigated. Association of 125I-ankyrin with KI-stripped inside-out erythrocyte membrane vesicles was found to proceed in two kinetic phases. The initial, fast phase (t1/2 approximately 15-30 min) involved predominantly the binding of ankyrin to low affinity sites (KD approximately 130 nM) in a pH-dependent manner. The apparent pKa values describing this reversible pH dependence (7.2 +/- 0.1 and 9.2 +/- 0.1) defined states of band 3 with high, moderate, and no capacity to bind ankyrin (in order of increasing pH). Since the cytoplasmic domain of band 3 also exists in 3 distinct conformational states characterized by apparent pKa values of 7.2 and 9.2, it was hypothesized that the reversible structural equilibrium in band 3 could influence ankyrin binding. The second or slow phase of ankyrin binding to band 3 involved the conversion of low to high affinity sites (KD approximately 13 nM). This phase, which was largely temperature and pH independent, required roughly an order of magnitude longer to reach completion than the fast phase. Unfortunately, even though the slow phase could be cleanly separated from the fast phase at low pH, insufficient data were available to formulate a physical interpretation of its origin. Significantly, however, even after completion of the slow phase under the most quantitative binding conditions identified, a maximum of only 26% of the band 3 was found to bind ankyrin in situ. Although higher ankyrin-band 3 stoichiometries may be achievable with the isolated cytoplasmic fragment of band 3, we interpret the above 1:4 stoichiometry to suggest that the tetramer of band 3 constitutes the predominant ankyrin binding oligomer of band 3 on the membrane.     

Viscoelasticity of the human red blood cell

We report here the first measurements of the complex modulus of the isolated red blood cell (RBC). Because the RBC is often larger than capillary diameter, important determinants of microcirculatory function are RBC deformability and its changes with pathologies, such as sickle cell disease and malaria. A functionalized ferrimagnetic microbead was attached to the membrane of healthy RBC and then subjected to an oscillatory magnetic field. The resulting torque caused cell deformation. From the oscillatory forcing and resulting bead motions, which were tracked optically, we computed elastic and frictional moduli, g' and g', respectively, from 0.1 to 100 Hz. The g' was nearly frequency independent and dominated the response at all but the highest frequencies measured. Over three frequency decades, g' increased as a power law with an exponent of 0.64, a result not predicted by any simple model. These data suggest that RBC relaxation times that have been reported previously, and any models that rest upon them, are artifactual; the artifact, we suggest, arises from forcing to an exponential fit data of limited temporal duration. A linear range of response was observed, but, as forcing amplitude increased, nonlinearities became clearly apparent. A finite element model suggests that membrane bending was localized to the vicinity of the bead and dominated membrane shear. While the mechanisms accounting for these RBC dynamics remain unclear, methods described here establish new avenues for the exploration of connections among the mechanical, chemical, and biological characteristics of the RBC in health and disease. storage modulus; loss modulus; magnetic twisting cytometry; erythrocyte; viscoelasticity; rheology     

Sodium movements in the human red blood cell

Measurements were made of the sodium outflux rate constant, ^o k _Na, and sodium influx rate constant, ⁱ k _Na, at varying concentrations of extracellular (Na_o) and intracellular (Na_c) sodium. ^o k _Na increases with increasing [Na_o] in the presence of extracellular potassium (K_o) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, ^o k _Na falls as [Na_o] rises from 0 to 6 mM; above 6 mM, ^o k _Na increases with increasing [Na_o]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Na_o] increases from 0 to 6 mM, ⁱ k _Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Na_c] increases, ^o k _Na at first increases and then decreases; this relation between ^o k _Na and [Na_c] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Na_c for Na_o via the Na-K pump mechanism.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Membrane rafts of the human red blood cell

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte’s, could be relevant also for other cell types.     

Elasticity of the human red blood cell skeleton

We have measured by optical tweezers micromanipulations the area expansion and the shear moduli of spectrin skeletons freshly extracted from human red blood cells, in different controlled salinity conditions. At medium osmolarity (150 mOsm/kg), we measure KC=9.7+/-3.4 microN/m, muC=5.7+/-2.3 microN/m, KC/muC=2.1+/-0.7. When decreasing the osmolarity, both KC and muC decrease, while KC/muC is nearly constant and equal to about 2. This result is consistent with the predictions made when modeling the spectrin skeleton by a two-dimensional triangular lattice of springs. From the measured elastic moduli we estimate the persistence length of a spectrin filament: xi approximately 2.5 nm at 150 mOsm/kg.     

ATP regulation of the human red cell sugar transporter

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.     

Permeability of the human red blood cell tomeso-erythritol

Summary Using¹⁴C-erythritol, we measured net as well as unidirectional erythritol fluxes. Up to near saturation, net and unidirectional fluxes were virtually identical and linearly related to the erythritol concentration in the medium (isotonic saline). No saturation of the transfer system was observed. At 20°C, a maximum of 60 to 70% of the erythritol flux could be inhibited by glucose, phlorizin, or a combination of both substances. Dinitrofluorobenzene and HgCl₂ also reduce erythritol permeability. These findings confirm the earlier conclusion of F. Bowyer and W. F. Widdas that the glucose transport system is involved in erythritol permeation. Glycerol partially inhibits the glucose-phlorizin-sensitive component of erythritol flux, but not the glucose-phlorizin-insensitive component. Apparently glycerol has a slight affinity to that portion of the glucose transport system which is involved in erythritol transfer, whereas the glucosephlorizin-insensitive fraction of erythritol movements is not identical with the glycerol system. This latter inference is supported by the observation that, in contrast to glycerol permeability, erythritol permeability is insensitive to variations of pH or to the addition of copper. The apparent activation energy of the glucose-phlorizin-sensitive and-insensitive fractions of erythritol permeation are 22.2 and 20.7 kcal/mole, respectively. These values are not significantly different from one another.     

Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.     

Trafficking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia caused by mutations in erythrocyte proteins including the anion exchanger, AE1 (band 3). This study examined seven missense mutations (L707P, R760Q, R760W, R808C, H834P, T837M, and R870W) located in the membrane domain of the human AE1 that are associated with this disease. The HS mutants, constructed in full-length AE1 cDNA, could be transiently expressed to similar levels in HEK 293 cells. Immunofluorescence, cell surface biotinylation, and pulse chase labeling showed that the HS mutants all exhibited defective cellular trafficking from the endoplasmic reticulum to the plasma membrane. Impaired binding to an inhibitor affinity matrix indicated that the mutant proteins had non-native structures and may be misfolded. Further characterization of the HS R760Q mutant showed no change in its oligomeric structure or turnover (half-life=15 h) compared to wild-type AE1, suggesting the mutant was not aggregated or targeted for rapid degradation via the proteasome. Intracellular retention of HS mutant AE1 would lead to destruction of the protein during erythroid development and would account for the lack of HS mutant AE1 in the plasma membrane of the mature red cell.     

Network-level analysis of metabolic regulation in the human red blood cell using random sampling and singular value decomposition

Background  

Extreme pathways (ExPas) have been shown to be valuable for studying the functions and capabilities of metabolic networks through characterization of the null space of the stoichiometric matrix (S). Singular value decomposition (SVD) of the ExPa matrix P has previously been used to characterize the metabolic regulatory problem in the human red blood cell (hRBC) from a network perspective. The calculation of ExPas is NP-hard, and for genome-scale networks the computation of ExPas has proven to be infeasible. Therefore an alternative approach is needed to reveal regulatory properties of steady state solution spaces of genome-scale stoichiometric matrices.     

The human red blood cell proteome and interactome

The red blood cell or erythrocyte is easily purified, readily available, and has a relatively simple structure. Therefore, it has become a very well studied cell in terms of protein composition and function. RBC proteomic studies performed over the last five years, by several laboratories, have identified 751 proteins within the human erythrocyte. As RBCs contain few internal structures, the proteome will contain far fewer proteins than nucleated cells. In this minireview, we summarize the current knowledge of the RBC proteome, discuss alterations in this partial proteome in varied human disease states, and demonstrate how in silico studies of the RBC interactome can lead to considerable insight into disease diagnosis, severity, and drug or gene therapy response. To make these latter points we focus on what is known concerning changes in the RBC proteome in Sickle Cell Disease.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.