Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Studies on lithium transport across the red cell membrane

Summary Ouabain-resistant Na⁺–Li⁺ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li⁺ for Na⁺ in a ratio of 11, was present in all four species. Li⁺ uptake by the exchange system increased 30-fold in the order man +–Na⁺ exchange in these species, but bears no relation to the Na⁺–K⁺ pump activity. The activity of the Na⁺–Li⁺ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li⁺ and Na⁺ were similar among the species and individuals (half saturation of the external site at about 1mm Li⁺ and 50mm Na⁺, respectively).50–60% of Na⁺–Li⁺ exchange was blocked by N-ethylmaleimide in all species.p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60–80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li⁺ unaltered. Phloretin was a potent inhibitor in all species. 1mm each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na⁺–Li⁺ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na⁺–Na⁺ exchange in beef erythrocytes.It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na⁺ for Na⁺, Na⁺ for Li⁺, or Li⁺ for Li⁺. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na⁺–K⁺ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na⁺ transport systems described in the literature.     

The temperature dependence of the facilitated transport ofd(+)-glucose across the human red cell membrane

Summary The rate of exit ofd(+)-glucose from human red cells was measured as a function of the extracellular glucose concentration over the temperature range 12 to 47°C. The results were analyzed at each temperature, according to the kinetic model of Widdas and of Rosenberg and Wilbrandt, in terms of the apparent maximum exit rate (V _max) and the apparent dissociation constant (K _m ) of the carrier-glucose complex. When the values ofV _max andK _m were obtained by the same graphical method as that used by Sen and Widdas, the results were very similar to theirs insofar as the effect of temperature is concerned. In particular, the apparent standard enthalpy of dissociation (H _m ) of the carrier-glucose complex does not vary with temperature, whereas the apparent activation energy (E _max) for the translocation of the carrier increases strongly with decreasing temperature. It is shown that the explanation of these findings given by Dawson and Widdas is internally inconsistent. Furthermore, the graphical method as used by these authors is unreliable at higher temperatures, whereK _m is large and consequently underestimatesK _m . An improved modification of the method, suggested by Bolis, Luly and Wilbrandt, overcomes this difficulty and leads to more reliable values forV _max andK _m . These new results show thatE _max decreases, and H _m increases, as the temperature is raised. This behavior is shown to be consistent with the modified kinetic model for sugar transport proposed by Wilbrandt, in which the translocation rate of the loaded carrier is assumed to be different from that of the empty carrier. The changes inE _max and H _m with temperature are the result of the difference in true activation energies for the translocation of the loaded and empty carrier.     

Anion transport across the red blood cell membrane mediated by dielectric pores

Summary The anion transport across the red blood cell membrane is assumed to occur by ionic diffusion through dielectric pores which are formed by protein molecules spanning the red blood cell membrane. The access of anions to the dielectric pores is regulated by anion adsorption sites positioned at the entrances of the pores. The adsorption of small inorganic anions to the adsorption sites is facilitated by ionizing cationic groups setting up a surface potential at the respective membrane surfaces. Applying the transition state theory of rate processes, flux equations for the unidirectional flux were derived expressing the unidirectional flux as a function of the fractional occupancies of anion adsorption sites at both membrane surfaces.The basic properties of the transport model were investigated. The concentration-dependence and the pH-dependence of the unidirectional fluxes were shown to depend upon the surface charge density and upon the affinity of the transported anion species to the anion binding sites. The concentration-response and the pH-response of the unidirectional fluxes of different anion species may differ substantially even if the anion species are transported by the same anion transport system. The model predicts a characteristic behavior of the Lineweaver-Burk plot and of the Dixon plot.A comparison between computer simulated and experimentally determined flux curves was made. By choosing a suitable set of parameters, the anion transport model is capable of simulating the concentration-dependencies and the pH-dependencies of the unidirectional sulfate and chloride flux. It is sufficient to change one single constant in order to convert the sulfate transport system into a chloride transport system. Furthermore, the model is capable of predicting the inhibitory action of chloride on the sulfate transport system. No attempts were made to fit the experimental data to the model. The behavior of the model was qualitatively in accordance with the experimental results.     

Kinetics of bicarbonate-chloride exchange across the human red blood cell membrane

The kinetics of bicarbonate-chloride exchange across the human red cell membrane was studied by following the time course of extracellular pH in a stopped-flow rapid-reaction apparatus during transfer of H+ into the cell by the CO2 hydration-dehydration cycle, under conditions where the rate of the process was determined by HCO3--Cl- exchange flux across the membrane. The flux of bicarbonate increased linearly with [HCO3-] gradient from 0.6 to 20 mM across the red cell membrane at both 37 degrees C and 2 degrees C, and decreased as transmembrane potential was increased by decreasing extracellular [Cl-]. An Arrhenius plot of the rate constants for the exchange indicates that the Q10 is strongly dependent on temperature, being about 1.7 between 24 degrees C and 42 degrees C and about 7 between 2 degrees C and 12 degrees C. These data agree well with the published values for Q10 of 1.2 between 24 degrees C and 40 degrees C and of 8 between 0 degrees C and 10 degrees C. The results suggest that different processes may determine the rate of HCO3- -Cl- exchange at low vs. physiological temperatures, and that the functional (and/or structural) properties of the red cell membrane vary markedly with temperature.     

The inactivation by fluorodinitrobenzene of glucose transport across the human erythrocyte membrane The effect of glucose inside or outside the cell

The inactivation of glucose transport in human red cells by fluorodinitrobenzene is accelerated by 120 mM glucose outside the cell but retarded at least 50% by 120 mM glucose inside the cell. This suggests that the transport system is predominantly in one conformation when there is glucose inside the cell, and in another conformation when there is glucose outside the cell.     

Calcium-calcium and calcium-strontium exchange across the membrane of human red cell ghosts

Summary Stationary and nonstationary state⁴⁵Ca fluxes as well as Sr–Ca exchange movements were studied in energy-depleted human erythrocyte ghosts at different intra-and extracellular Ca concentrations. Influx and efflux followed the kinetics of a closed two-compartment system. The influx and efflux rate constants (k _in andk _out, respectively, fractions of total extra- or intracellular⁴⁵Ca that move in one direction per unit time) were similar in magnitude. They decreased with increasing Ca concentration on the cisside and increased with increasing Ca concentration on the trans-side of the membrane. Hence, the fluxes in both directions were characterized by saturation kinetics and appeared to be partially caused by an exchange diffusion mechanism. In the presence of a moderate inward (up to 8mm) or outward (up to 2mm) Ca concentration gradient, k_in andk _out did not vary in the course of an experiment and did not differ significantly from rates which were measured under stationary state conditions. Extracellular Sr induced an outward transport of intracellular Ca against the concentration gradient (counter-transport). The resulting inward Ca concentration gradient (maximal inside-to-outside concentration ratio as 1 to 3) persisted since extra- and intracellular Sr did not equilibrate. Analogous results were obtained studying⁴⁵Ca–⁴⁰Ca countertransport. In net flow experiments Ca–Sr exchange proved to occur on a one-for-one basis. Ca–Sr exchange was additive to the noncoupled Ca and Sr net downhill movements. The experimental results suggest that a specific ATP-independent Ca transfer system exists in the erythrocyte membrane which acts symmetrically on the two sides of the membrane and is restricted to a tightly coupled one-for-one exchange diffusion.     

Kinetic characteristics of bicarbonate-chloride exchange across the neonatal human red cell membrane

The kinetics of HCO₃^?/Cl^? exchange across red cell membrane of newborn infants was studied using a stopped-flow rapid reaction apparatus with a glass pH electrode attached. The measured apparent permeability P is (1.35±0.08 (S.E.)) · 10^?4 cm/s (n=30) for newborns, compared with (3.1 ± 0.4) · 10^?4 cm/s (n=15) for adults. These correspond to half-times of 0.2 s for newborns and 0.1 s for adults indicating that neonatal red cells exchange Cl^? for HCO₃^? only half as fast as do adult cells. The temperature dependence of the exchange rate was studied from 2 to 42°C. From the Arrhenius plot the activation energy of the exchange process in neonatal red cells changes from 22.9 kcal/mol (low temperature) to 4.8 kcal/mol (physiological temperature) at a transition temperature of 17°C. These values are lower than the corresponding values for adult red cells, 34.7 and 10.2 kcal/mol. HCO₃^?/Cl^? exchanges in both adult and neonatal red cells are inhibited by phlorizin. Inhibition constants K_i are 0.8 mM and 2.5 mM for adults and newborns, respectively. The differences in the values of the HCO₃^?/Cl^? exchange rate constant and the activation energy of the exchange process between neonatal and adult red cells indicate that there is a modification of HCO₃^?/Cl^? transport system in the neonatal red cell membranes.     

Faster lactate transport across red blood cell membrane in sickle cell trait carriers.

The physical and physiological behavior of sickle cell trait carriers (AS) is somewhat equivocal under strenuous conditions, although this genetic abnormality is generally considered to be a benign disorder. The occurrence of incidents and severe injuries in AS during exercise might be explained, in part, by the lactic acidosis due to a greater lactate influx into AS red blood cells (RBCs). In the present study, the RBC lactate transport activity via the different pathways was compared between AS and individuals with normal hemoglobin (AA). Sixteen Caribbean students, nine AS and seven AA, performed a progressive and maximal exercise test to determine maximal oxygen consumption. Blood samples were obtained at rest to assess haematological parameters and RBC lactate transport activity. Lactate influxes [total lactate influx and monocarboxylate transporter (MCT-1)-mediated lactate influx] into erythrocytes were measured at four external [14C]-labeled lactate concentrations (1.6, 8.1, 41, and 81.1 mM). The two groups had similar maximal oxygen consumption. Total lactate influx and lactate influx via the MCT-1 pathway were significantly higher in AS compared with AA at 1.6, 41, and 81.1 mM. The maximal lactate transport capacity for MCT-1 was higher in AS than in AA. Although AS and AA had the same maximal aerobic physical fitness, the RBCs from the sickle cell trait carriers took up more lactate at low and high concentrations than the RBCs from AA individuals. The higher MCT-1 maximal lactate transport capacity found in AS suggests greater content or greater activity of MCT-1 in AS RBC membranes.     

Evidence of active transfer of certain non-electrolytes across the human red cell membrane

1. Permeability of the human erythrocyte to glycerol, as indicated by the course of hemolysis and volume changes, is depressed by Cu(++), Hg(++), I(2), p-chloromercuribenzoate, and phlorhizin, without effecting general permeability changes. In so far as tested (Cu(++), p-ClHgB), these inhibitors delay exit of glycerol from the cell as well as its entry. 2. Permeability to glucose is similarly depressed by I(2) and phlorhizin, and is extremely sensitive to Hg(++) and p-chloromercuribenzoate, but is not affected by Cu(++). An extensive series of other enzyme poisons is without effect in either system. 3. The effects of the sulfhydryl inhibitors are prevented or reversed in the presence of glutathione, cysteine, etc. 4. The kinetics of the volume changes in glucose-saline solutions indicates a mechanism for transport of glucose into the cell, regulated by the existing intracellular concentration, rather than by simple diffusion gradients. 5. The intermediation of a sulfhydryl group at the cell surface, probably an enzymatic phosphorylation, is suggested as an essential step in the passage of glycerol, glucose, and other like substances, across the human red cell membrane.     

Anion transport across the red blood cell membrane and the protein in band 3.

The paper reviews existing evidence for the participation of the protein in band 3 (nomenclature of Steck, [1]) in anion transport across the red cell membrane and discusses the possible role of common binding sites on band 3 for 1-fluoro-2,4-dinitrobenzene, 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid and dihydro 4,4'-diisothiocyanato stilbene-2,2'-disulfonic acid in the transport process.     

New evidence for the essential role of arginine residues in anion transport across the red blood cell membrane

2,3-Butanedione, in the dark and in the presence of borate, reacts rapidly to inactivate the sulfate equilibrium exchange across the human red cell membrane. Reactivation occurs spontaneously after the removal of borate, indicating the reaction of butanedione with essential arginine residues. The inactivation of the transport system depends on the concentration of the reagent, on the incubation time and exhibits pseudo-first-order kinetics. Chloride ions are able to protect the transport system against inactivation with the reagent. This would suggest the participation of the modified residue in the substrate binding site. When the transport system is inhibited to 50-60% by butanedione, the transporter can still bind covalently the anion transport inhibitor 2H2DIDS up to 85 +/- 12% of its total binding capacity. 3H2DIDS concentration was either 3.15, 10 or 20 microM. Modification of resealed ghosts with 50 mM butanedione under conditions where the transport system is to more than 75% inhibited, causes a reduction of only about 30% of the reversibly bound 3H2DIDS.