Analysis of the pH dependence of folate binding and transport by rat kidney brush border membrane vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

Transmembrane pH gradients and functional heterogeneity in reconstituted vesicle systems

The pH-sensitive, membrane impermeant fluorescence probes 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine; pK_a = 7.2) and 1-naphthol-3,6-disulfonate (Naps pK_a = 8.2) can be simultaneously entrapped within the intravesicular aqueous compartment of unilamellar vesicles and reconstituted proteoliposomes, where they function as reliable reporters of the intravesicular pH. Because the two probes are sensitive to pH over different but overlapping ranges, the useful monitoring range for the co-trapped probe pair extends from pH 6.5 to 9. In vesicles pre-equilibrated at a given pH and then subjected to a sudden change in external pH, the rate and extent of the subsequent change in internal pH are identical at all times during the re-equilibration, regardless of which probe is used to monitor the change. However, in reconstituted bacteriorhodopsin proteoliposomes, the size of the transmembrane pH gradient generated in the light always appears greater when pyranine is used to monitor internal pH. This discrepancy can most readily be understood in terms of heterogeneity in the vesicle suspension, with at least two populations of vesicles, one active in proton and one inactive. A simple algorithm was developed which generates, from the observed internal pH changes for two probes of different pK_a, the percentage of vesicles which are inactive, as well as the actual internal pH of the active fraction. The applicability of this algorithm was subsequently confirmed using a suspension of vesicles in which the level of heterogeneity was deliberately altered by the addition of various amounts of gramicidin. The apparent transmembrane pH gradient for the vesicle population as a whole decreased with increasing gramicidin, as did the calculated percentage of vesicles able to maintain a pH gradient, while the transmembrane gradient calculated for the active vesicle fraction only was essentially unaffected by gramicidin.     

Protonmotive force and catecholamine transport in isolated chromaffin granules.

The effect of the transmembrane potential (delta psi) and the proton concentration gradient (delta pH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in isolated bovine chromaffin granules. Freshly isolated chromaffin granules had an intragranular pH of 5.5 as measured by [14C]methylamine distribution. The addition of ATP to a suspension of granules resulted in the generation of a membrane potential, positive inside, as measured by [14C]thiocyanate (SCN-) distribution. The addition of carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton translocator, resulted in a reversal of the potential to negative values (measured by [3H]tetramethylphenylphosphonium (TPMP+)) approaching -90 mV. Changing the external pH of a granular suspension incubated with FCCP produced a linear perturbation in the measured potential from positive to negative values, which can be explained by the distribution of protons according to their electrochemical gradient. When ammonia (1 to 50 mM) was added to highly buffered suspensions of chromaffin granules there was a dose-dependent decrease in the transmembrane proton gradient (delta pH) and an increase in the membrane potential (delta psi). On the other hand, thiocyanate or FCCP, at varying concentration, produced a dose-related collapse of the membrane potential and had no effect upon the transmembrane proton gradient. The addition of larger concentrations of catecholamines caused a decrease in the transmembrane proton gradient and an increase in the membrane potential. Time-resolved influx of catecholamines into the granules was studied radiochemically using low external catecholamine concentrations. The accumulation of epinephrine or norepinephrine was over one order of magnitude greater in the presence of ATP than in its absence. The rate and extent of amine accumulation was found to be related to the magnitude of the membrane potential at fixed transmembrane proton concentration (delta pH) values. Likewise, the accumulation was related to the magnitude of the delta pH at fixed membrane potential values. These results suggest that the existence of both a transmembrane proton gradient and a membrane potential are required for optimal catecholamine accumulation to occur.     

Energy transduction in Escherichia coli: new mutation affecting the Fo portion of the ATP synthetase complex.

A mutation affecting the intrinsic membrane portion (BFo) of the ATP synthetase complex is described. The phenotype is different from previously reported BFo mutants. This mutation results in the ability of membranes lacking the extrinsic membrane portion (BF1) of the ATP synthetase complex to maintain a transmembrane pH gradient. Unlike other BFo mutants, this strain, NR71, is capable of utilizing ATP hydrolysis for the formation of a transmembrane pH gradient.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

Cation gradient dependence of the steps in thrombin stimulation of human platelets

Thrombin causes a dose-dependent depolarization of the transmembrane potential of normal human platelets which can be continuously measured by the fluorescent probe, 3,3'-dipropylthiodicarbocyanine, whose distribution across the plasma membrane has been shown to be dependent upon the membrane potential. The dose-dependent depolarization of the platelet's negative membrane potential by thrombin is in large part due to a rapid uptake of sodium. Both the membrane potential change and the rapid sodium influx can be inhibited by a fast acting analog of amiloride, a sodium channel blocker, while valinomycin, a potassium ionophore, has no effect on the potential change nor on the sodium uptake, suggesting that the transmembrane potassium gradient is not important in the thrombin-induced depolarization. Neither the secretion of serotonin nor that of lysosomal enzymes nor the secondary release of the fluorescent probe which correlates with the lysosomal enzyme secretion occur if treatment with valinomycin precedes activation by thrombin. It is thus apparent that: 1) the change in the membrane potential induced by thrombin is directly dependent upon the transmembrane sodium gradient and is primarily due to a dose-dependent sodium uptake by the platelets; and 2) the thrombin-induced secretory processes are dependent upon maintenance of the transmembrane potassium gradients.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

A variable stoichiometry model for pH homeostasis in bacteria.

The composition of the proton-motive force of a hypothetical bacterial cell of wide pH tolerance is analyzed according to a model whereby the electron transport chain and various proton-linked sodium and potassium ion transporting modes are responsible for the development of the membrane potential and the chemical potentials of the three cations. Simultaneous use of two or more modes employing the same metal cation, but at a different stoichiometric ratio with respect to protons, produces nonintegral stoichiometry; the modes could represent either different devices or different states of a single device. Cycling of the cation, driven by proton-motive force, results. The relative conductances of the various modes are postulated to be pH-dependent. The pattern of potentials that results is qualitatively in accord with current knowledge and may reflect the mechanism of pH homeostasis in bacteria. The membrane potential is outwardly directed (positive inside) at extremely acid pH, becoming inwardly directed as the pH increases; the pH gradient across the membrane is large and inwardly directed (alkaline inside) at acid pH, becoming smaller and eventually inverting at alkaline pH values; the transmembrane potassium gradient is outwardly directed (high concentration inside) at all pH values; the transmembrane sodium gradient is inwardly directed at all pH values, following the pH gradient from acid through neutral pH, but then diverging at alkaline pH.     

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.     

Estimation of transmembrane potentials from phase equilibria of hydrophobic paramagnetic ions.

Positively charged hydrophobic spin labels have been synthesized which respond to transmembrane potentials in sonicated liposomes. Electron paramagnetic resonance spectroscopy is used to show that the distribution of these probes between aqueous and membrane phases changes as a function of transmembrane potential. When liposomes are made more inside-negative, the fraction of membrane associated probe increases while the fraction of probe in the aqueous phase decreases. The results are in quantitative agreement with a simple equilibrium thermodynamic theory which allows estimation of absolute transmembrane potentials in phospholipid vesicles.     

Role of a transbilayer pH gradient in the membrane fusion activity of the influenza virus hemagglutinin: Use of the R18 assay to monitor membrane merging

It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferro-oxidans.

Thiobacillus ferro-oxidans is capable of using the oxidation of Fe2+ by O2 at pH 2.0 as the sole source of energy for growth and CO2 fixation. The bacterium maintains an intracellular pH of 6.5 over a range of external pH from 1.0 to 8.0, as measured by [14C]acetate and [3H]methylamine distribution. The membrane potential was estimated by the distribution of the lipid-soluble cation dibenzyldimethylammonium and the anion SCN-. At pH 2.0 (the pH of growth) during Fe2+ oxidation the transmembrane pH gradient is 4.5 units with an opposing membrane potential of -10mV, giving a proton electrochemical gradient of +256mV. This gradient is actively maintained.     

Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli.

A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.     

Sensitivity of erythrocyte acethylcholinesterase to inhibition by linolenoyl sorbitol. Dependence on a transmembrane potential.

Acetylcholinesterase activity of human erythrocytes is known to be inhibited by linolenoyl sorbitol, the inhibition being critically dependent on cell membrane intactness. The extent of enzyme inhibition by the added lipid is correlated with the magnitude of Cl- gradient across the erythrocyte membrane, indicating that enzyme sensitivity is associated with a transmembrane potential. If linolenoyl sorbitol is allowed to interact with the erythrocytes while a Cl- gradient exists, enzyme sensitivity can subsequently be demonstrated not only in the absence of a gradient but even when the cells are lyzed. It is consluded that the transmembrane potential determines the accessibility of a membrane component to the added lipid.     

Formation of the transmembrane electrochemical potential on Staphylococcus cells and membrane vesicles

The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]