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

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, 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 $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · 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 $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ 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.     

Quantitative predictions of a noncarrier model for glucose transport across the human red cell membrane

There is an increasing amount of experimental data on transport across biological membranes which cannot be readily accommodated by classical mobile carrier models. We propose models for membrane transport based upon current concepts in molecular enzymology, in which the membrane component involved in transport is an oligomeric protein which undergoes substrate-induced conformational changes. A number of paradoxical observations on glucose transport in the human erythrocyte are explained if the protein involved is a tetramer possessing two classes of binding sites with different affinities for glucose. We develop in detail a particular model of this type, the internal transfer model, in which transport occurs by transfer of substrate from one subunit to another of the protein. The fit of the predictions of the internal transfer model with most of the experimental data is very good. Those data which cannot be fitted by the model cannot be accounted for by any presently available model. We extend our model qualitatively to include the sodium-activated cotransport systems for sugars and amino acids.     

Lanthanide-stimulated glucose and proline transport across rabbit intestinal brush-border membranes

Trivalent cations of the lanthanide series (La3+----Yb3+) stimulated uptake of proline or glucose in rabbit small intestinal brush-border membrane vesicles. The lanthanides stimulated uptake to an extent greater than Al3+, choline, and in many cases, Na+. A time-course of Er3+-stimulated glucose uptake gave initial rates and overshoots greater than Na+ stimulation. The best activators were Sm3+, Eu3+ and Tm3+, which stimulated proline initial uptakes by 400-600%, and stimulated glucose uptake by 120-150%, compared to Na+. The best lanthanide cotransport activators possessed high third ionization potentials.     

Mechanisms for the facilitated diffusion of substrates across cell membranes

Two classes of theoretical mechanisms for protein-mediated, passive, transmembrane substrate transport (facilitated diffusion) are compared. The simple carrier describes a carrier protein that exposes substrate influx and efflux sites alternately but never both sites simultaneously. Two-site models for substrate transport describe carrier proteins containing influx and efflux sites simultaneously. Velocity equations describing transport by these mechanisms are derived. These equations take the same general form, being characterized by five experimental constants. Simple carrier-mediated transport is restricted to hyperbolic kinetics under all conditions. Two-site carrier-mediated transport may deviate from hyperbolic kinetics only under equilibrium exchange conditions. When both simple- and two-site carriers display hyperbolic kinetics under equilibrium exchange conditions, these models are indistinguishable by using steady-state transport data alone. Seven sugar transport systems are analyzed. Five of these systems are consistent with both models for sugar transport. Uridine, leucine, and cAMP transport by human red cells are consistent with both simple- and two-site models for transport. Human erythrocyte sugar transport can be modeled by simple- and two-site carrier mechanisms, allowing for compartmentalization of intracellular sugars. In this instance, resolution of the intrinsic properties of the human red cell sugar carrier at 20 degrees C requires the use of submillisecond transport measurements.     

Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes

Summary After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4-diisothiocyano-2,2-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976,Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.This paper is dedicated to the memory of Walther Wilbrandt.     

Some aspects of zinc transport across eel (Anguilla anguilla) red blood cell membranes.

Zinc movement across eel and human red blood cell membranes was measured by atomic absorption spectrophotometry. It was observed that:

• 1) In human red blood cells zinc uptake is twice as rapid as in fish red blood cells over a temperature range of 10-40°C. The low rate of zinc uptake in eel red blood cell may be simply the side effect of different surface area to volume ratios for the differences in cell size or, it may be due to the low permeability of bicarbonate through the red blood cell membranes.
• 2) Zinc uptake measured in eel and human red blood cells treated and untreated with external trypsin shows different features. The zinc uptake was reduced by about 40% in treated eel red blood cells with respect to the total uptake of untreated red blood cells. Human red blood cells treated and untreated with trypsin do not show any differences in the amount of zinc transported.
• 3) In fish red blood cells, zinc uptake in NANO₃ medium is markedly reduced, compared with that measured in NaCl medium. The [Zn²⁺_i slightly increases in the presence of bicarbonate. In human red blood cells in NANO₃ medium the zinc uptake is strongly reduced and the presence of bicarbonate marginally increases the zinc influx.
• 4) In eel red blood cells there seem to be two independent pathways for zinc uptake: one DIDS-sensitive and the other DIDS-insensitive. DIDS 10 μM inhibits only 64% of the total zinc transported. Iincreasing the DIDS concentration did not give more inhibition. In human red blood cells only one DIDS-sensitive pathway for zinc transported seems to exist, because, 2,5 μM DIDS inhibits 97% of zinc uptake.

     

Vanadate inhibition of active Ca2+ transport across human red cell membranes

(1) Vanadate (pentavalent vanadium) inhibits with high affinity ($\text{K}{}_{0.5}\text{= 3 μ}\text{M}$) the ATP-dependent Ca²⁺ efflux in reconstituted ghosts from human red cells. (2) To inhibit Ca²⁺ efflux vanadate has to have access to the inner surface of the cell membrane. (3) The inhibitory effect of vanadate is potentiated by intracellular Mg²⁺ and by intracellular K⁺. (4) Ca²⁺ in the external medium antagonizes the inhibitory effect of vanadate.     

Biomimetic polyesters and their role in ion transport across cell membranes

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.     

Kinetics of bicarbonate and chloride transport in human red cell membranes

Unidirectional [14C]HCO3- and 36Cl- efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0 degrees C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that 14CO2 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with CAn constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(eff) = J(eff)max.C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant Ki mM) to an intracellular site was included in the model (MS fit): J(eff) = J(eff)max.C(i)/[(K1/2 + C(i)).(1 + C(i)/Ki)]. The MM fits show that the external half-saturation constant, Ko1/2 ( = C(o)An for J(eff,o) = 1/2.j(eff,o)max) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM Ko1/2 is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(eff,o)max (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, Ki1/2 mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(eff,i)max (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO-3) and 0.50 (Cl-). The half-inhibition constants of the MS fit, Ki, are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, Ks1/2, is 20.2 (HCO-3) and 23.9 (Cl-) mM, and J(eff,s)max is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM Ks1/2 is 30.4 nM (HCO3-) and 50.1 mM (Cl-), and Ki is 541 mM (HCO3-) and 392 mM (Cl-).(ABSTRACT TRUNCATED AT 400 WORDS)     

A method for measuring anion transfer across red cell membranes by continuous monitoring of fluorescence

A method was developed for the identification of Ca²⁺-binding proteins after electrophoresis on polyacrylamide gels. The method involves equilibration of the gel with ⁴⁵Ca either during or after electrophoresis, followed by visualization of the ⁴⁵Ca-binding proteins by autoradiography.     

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.