Interactions of divalent cations or basic proteins with phosphatidylethanolamine vesicles

Small unilamellar vesicles have been prepared from phosphatidylethanolamine by sonication of the lipid in aqueous buffers of low ionic strength and high pH. These vesicles and their interactions with various di- and trivalent cations have been characterized using freeze-fracture electron microscopy. Phosphatidylethanolamine from 4 sources was examined: Hens' yolk phosphatidylethanolamine, human grey matter phosphatidylethanolamine, Escherichia coli phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine. The phosphatidylethanolamine from natural sources formed spherical, uniform 20–40 nm vesicles while dimyristoyl phosphatidylethanolamine formed larger, 70 × 25 nm, disc-shaped vesicles when sonicated above the phase transition temperature. Fusion of the unilamellar egg phosphatidylethanolamine, E. coli phosphatidylethanolamine and human grey matter phosphatidylethanolamine vesicles was induced by dialysis against buffers containing 2.0 nM Ca⁺ or 3.0 mM Mg²⁺. The fusion of the vesicles resulted in the precipitation of the lipid and the formation of multilamellar and, in some cases, hexagonal II structures. Dimyristoyl phosphatidylethanolamine vesicles were precipitated at 55°C by 1.0 mM Ca⁺ or 2.0 mM Mg²⁺. Treatment of the calcium- and magnesium-precipitated vesicles of hen's egg yolk phosphatidylethanolamine, E. coli phosphatidylethanolamine, human grey matter phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine with EDTA resulted in resuspension of the lipid. The specific size and shape of the vesicles formed in this manner depends on the type of phosphatidylethanolamine and ion involved. Dialysis of the Ca⁺- and Mg²⁺-precipitated egg phosphatidylethanolamine vesicles against buffer containing no Ca⁺, Mg²⁺ or EDTA also resulted in dissociation of the precipitate and formation again of a new vesicle population. This evidence indicates that the Ca⁺ and Mg²⁺ are not strongly bound to the phosphatidylethanolamine.Egg phosphatidylethanolamine vesicles would fuse in the presence of many di- and trivalent ions. Egg phosphatidylethanolamine vesicles were precipitated by beryllium, aluminum, chromium, manganese, cobalt, nickel, copper, zinc, strontium, cadmium, barium, lanthanium, mercury and lead. The amount of ion required to precipitate the vesicles and the type of structure resulting from the fusion of the vesicles was found to be unique for each ion.Small unilamellar vesicles prepared from egg phosphatidylethanolamine were reacted with several basic proteins (cytochrome c, basic protein from human myelin, protamine, poly-l-lysine and cationically-modified ferritin). The basic proteins also initiated the fusion of egg phosphatidylethanolamine vesicles but these proteins did not fuse egg phosphatidylcholine vesicles nor did normal ferritin initiate fusion. Human myelin basic protein initiated the fusion of dimyristoyl phosphatidylethanolamine vesicles above and below the phase transition of this lipid.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose – influence of α-tocopherol, β-carotene,zeaxanthin and cholesterol

The permeability of large unilamellar vesicles formed from digalactosyldiacylglycerol for glucose and protons was measured. The vesicle composition was modified by addition of different terpenoids: α-tocopherol, cholesterol, zeaxanthin and β-carotene. The digalactosyldiacylglycerol species composition was dominated by the species 18:2/18:2 and 18:1/18:2. Using the self-quenching properties of the fluorescent probe 6-carboxyfluorescein trapped in the aqueous space of the vesicles, the permeability for glucose was determined with a glucose gradient of 800 mM. The calculated permeability coefficient for glucose was in the range of 4.85.10^–10 to 1.12.10^–9 cm.s^–1. For proton permeability measurements, the pH-sensitive fluorescent dye pyranine was used. The proton permeability was measured with a pH gradient of 0.6 pH units from 7.0 to 7.6 with valinomycin present to dissipate any diffusion potential across the membrane. The permeability coefficients for protons were in the range of 3.5.10^–8 to 1.0.10^–7 cm.s^–1. Digalactosyldiacylglycerol vesicles with 5 % α-tocopherol or 10 % cholesterol or 2 % zeaxanthin reduced the permeability for protons, the two latter significantly as compared to digalactosyldiacylglycerol vesicles. α-Tocopherol (5 %) decreased the permeability for glucose remarkably and so did cholesterol (10 %). β-Carotene (< than 1 %) and zeaxanthin (2 %) in galactolipid vesicles, however, increased the permeability. The significance of these results is discussed in relation to the physiological functions of galactolipids and terpenoids in chloroplast membranes.     

Thallium interaction with the gastric (K,H)-ATPase

Summary The gastric (K,H)-ATPase has been shown to catalyze an electroneutral H⁺ for K⁺ exchange. Tl⁺ is able to substitute for K⁺ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 m as compared to about 870 m for K⁺. The ability of Tl⁺ to participate in transport is shown by the development of pH gradients in the presence of Tl⁺ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl⁺ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl⁺ concentrations above 3.0mm. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl⁺ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl⁺ concentrations, following the initial Tl⁺ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl⁺ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t _1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl⁺ concentrations in excess of 1 to 4mm are able to dissipate intravesicular proton gradients by an electrically coupled H⁺ for Tl⁺ exchange. Thus, although Tl⁺ is able to activate the gastric ATPase by mimicking K⁺, the permeability of this cation in lipid bilayers tends to uncouple H⁺ transport at concentrations high enough to generate detectable proton gradients.     

Passive H+/OH− permeability in epithelial brush border membranes

Passive H⁺/OH^– permeability across epithelial cell membranes is rapid and leads to partial dissipation of H⁺/OH^– gradients produced by H⁺ pumps and ion gradient-coupled H⁺/OH^– transporters. A heterogeneous set of H⁺/OH^– transport mechanisms exist in biological membranes: lipid solubility/diffusion, protein-mediated transport by specific proteins or by slippage through ion-coupled H⁺/OH^– transporters, and transport at the protein/lipid interface or through protein-dependent defects in the lipid structure. A variety of methods are available to study protein transport mechanisms accurately in cells and biomembrane vesicles including pH electrode recordings, pH-sensitive fluorescent and magnetic resonance probes, and potentiometric probes. In brush border vesicles from the renal proximal tubule, the characteristics of passive H⁺/OH^– permeability are quite similar to those reported for passive H⁺/OH^– permeability through pure lipid bilayers; slippage of protons through the brush border Na⁺/H⁺ antiporter or through brush border water channels is minimal. In contrast, passive H⁺/OH^– permeability in brush border vesicles from human placenta is mediated in part by a stilbene-sensitive membrane protein. To demonstrate the physiological significance of passive renal brush border H⁺/OH^– transport, proximal tubule acidification and cell pH regulation mechanisms are modeled mathematically for states of normal and altered H⁺/OH^– permeabilities.     

Effects of temperature on the phase behavior and permeability of thylakoid lipid vesicles : relevance to chilling stress

Large unilamellar vesicles composed of thylakoid glycolipids, phosphatidylglycerol, and varying proportions of dipalmitoylphosphatidylglycerol (DPPG) have been examined for the temperature dependence of their permeability to ⁸⁶Rb⁺ and for the occurrence of liquid-crystalline to gel (L_α-to-L_β) phase separations. In vesicles in which the normal 12 mole percent of moderately unsaturated thylakoid phosphatidylglycerol was partially or completely replaced by DPPG, analysis by differential scanning calorimetry indicated that an L_α-to-L_β phase separation did not occur between 0 and 60°C. However, in similar vesicle dispersions that were first subjected to a freeze-thaw cycle, L_α-to-L_β phase separations were observed to occur between 17 and 53°C. The temperature and enthalpy of these phase separations were closely related to the proportion of DPPG in the original lipid mixture. In parallel experiments, large unilamellar vesicles were measured for their permeability to ⁸⁶Rb⁺ between 7 and 30°C. There were no systematic increases in permeability to ⁸⁶Rb⁺ as a function of DPPG content at the temperatures relevant to chilling stress in higher plants. It is concluded that (a) L_α-to-L_β phase separations do not occur in well-defined galactolipid vesicles containing ≤12 mole percent DPPG between 0 and 60°C and (b) these vesicles show no alterations in permeability to ⁸⁶Rb⁺ between 7 and 30°C that are relevant to chilling stress in higher plants.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca²⁺ in the micromolar range. Optimal activity is reached at a free Ca²⁺ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with ⁴⁵Ca²⁺, ⁸⁶Rb⁺, or ²²Na⁺, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb⁺ and Na⁺ was measured, when the vesicles were pretreated with ATP and Mg²⁺.     

Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles

Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H⁺-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H⁺ was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H⁺-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K_m for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H⁺-ATPase activity in vivo.     

Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu³, which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe’s pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe’s pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu³ was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu³ was found to be superior to the commercially available pH indicators.     

Proton translation in submitochondrial vesicles

An investigation of proton translocation in submitochondrial vesicles from rat liver has been made under simple experimental conditions. Choline chloride was used both as the oxidizable substrate and the ionic medium for the measurement of activity during oxygen pulse experiments:

1. The passive permeability measured from the decay of proton efflux after an oxygen pulse could be described by a first-order equation. An H⁺/O ratio of 2·5 was obtained for choline oxidation in the presence of oligomycin and/or MgCl₂. Oligomycin decreased the passive proton permeability and respiration, concomitant with an increase in proton uptake. Respiratory control was directly related to the passive proton permeability and inversely related to the magnitude of the proton gradient. The decreased respiration and passive permeability reflecting respiratory control is most evident in the pH rang 5·8–7·5.
2. Preparation of submitochondrial vesicles in the presence of EDTA resulted in proton production during an oxygen pulse given at alkaline pH. Cytochromec enhanced proton uptake by approximately 1 H⁺/cytochromec, but only in the presence of Triton X-100. These results are indicative of the asymmetric behavior of the coupling membrane and provide direct evidence of the participation of electron transport components in proton translocation.

     

The effects of α-synuclein on phospholipid vesicle integrity: a study using 31P NMR and electron microscopy

Associations between the 140 amino acid protein α-synuclein (asyn) and presynaptic vesicles may play a role in maintaining synaptic plasticity and neurotransmitter release. These physiological processes may involve disruption and fusion of vesicles, arising from interactions between specific regions of asyn, including the highly basic N-terminal domain, and the surface of vesicles. This work investigates whether asyn affects the integrity of model unilamellar vesicles of varying size and phospholipid composition, by monitoring paramagnetic Mn²⁺-induced broadening of peaks in the ³¹P nuclear magnetic resonance spectrum of the lipid head groups. It is shown that asyn increases the permeability to Mn²⁺ of both large (200nm diameter) and small (50nm diameter) vesicles composed of zwitterionic phosphatidylcholine and anionic phosphatidylglycerol at protein/lipid molar ratios as low as 1:2000. Further experiments on peptides corresponding to sequences in the N-terminal (10–48), C-terminal (120–140) and central hydrophobic (71–82) regions of asyn suggest that single regions of the protein are capable of permeabilizing the vesicles to varying extents. Electron micrographs of the vesicles after addition of asyn indicate that the enhanced permeability is coupled to large-scale disruption or fusion of the vesicles. These results indicate that asyn is able to permeabilize phospholipid vesicles at low relative concentrations, dependent upon the properties of the vesicles. This could have implications for asyn playing a role in vesicle synthesis, maintenance and fusion within synapses.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

In?Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H⁺/Na⁺ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na⁺ and H⁺ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na⁺ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na⁺-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na⁺ transport and electrogenic H⁺ transport, respectively, in the presence and absence of Na⁺. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na⁺ transport by IAR requires Na⁺ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H⁺/Na⁺ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.     

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

NaCl Induces a Na/H Antiport in Tonoplast Vesicles from Barley Roots

Evidence was found for a Na⁺/H⁺ antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg²⁺, and the pH gradient gradually dissipated. When 50 millimolar K⁺ or Na⁺ was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K⁺ to vesicles from salt-grown roots. However, when 50 millimolar Na⁺ was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K⁺ and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na⁺/H⁺ exchange. The Na⁺/H⁺ exchange rate for vesicles from salt-grown roots showed saturation kinetics with respect to Na⁺ concentration, with an apparent K_m for Na⁺ of 9 millimolar. The rate of Na⁺/H⁺ exchange with 10 millimolar Na⁺ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.     

Na+, K+ and Cl− selectivity of the permeability pathways induced through sterol-containing membrane vesicles by amphotericin B and other polyene antibiotics

Membrane diffusion potentials induced by amphotericin B (AmB), amphotericin B methyl ester (AmE), N-fructosyl AmB (N FruAmB) and vacidin, an aromatic polyene antibiotic, in ergosterol- or cholesterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUV), were measured in various media, in order to determine the relative selectivity of Na⁺, K⁺, Cl^– and other ions in these environments. Changes in the membrane potential were followed by fluorescence changes of 3,3-dipropylthiadicarbocyanine (diS-C₃-(5)). Subtle changes in intercationic selectivity were monitored by measuring biionic potentials, using the fluorescent pH sensitive probe pyranine. In all the cases studied, the intereationic selectivity of the permeability pathways induced by the four antibiotics was weak compared to that of specific biological channels, though distinct differences were noted. With AmB the selectivity appeared to be concentration dependent. Above 5 × 10^–7 M, the sequence determined for sterol-free small unilamellar vesicles (SUV) and cholesterol-containing SUV and LUV, Na⁺ > K⁺ > Rb⁺ Cs⁺ > Li⁺ (sulfate salts), corresponded closely to Eisenman selectivity sequence number VII. At 5 × 10^–7 M and below the selectivity switched from Na⁺ > K⁺ to K⁺ > Na ⁺. In contrast, Li⁺ was the most permeant ion for AmB channels in the presence of ergosterol. The selectivity between Na⁺ or K⁺ vs. Cl^– varied with the antibiotic. It was very strong with vacidin at concentrations below 5 × 10^–7 M, smaller with AmB, nil with AmE and N FruAmB. The selectivities observed were antibiotic, concentration and time de pendent, which confirms the existence of different types of channels.Abbreviations AmB amphotericin B - AmE amphotericin B methylester - BLM bilayer membranes - DiSC₃(5) 3,3-dipropylthi-acarbocyanine iodide - DMSO dimethylsulfoxide - EPC egg yolk lecithin - FCCP carbonyl cyanide p-trifluoro methoxyphenyl-hydrazone - HEPES N-(2-hydroxyethylpiperazine)-N-(2-ethanesulfonic acid) - LUV large unilamellar vesicles - MOPS 3-(N-morpholino)propanesulfonic acid - N-Fru AmB N(1-deoxy-D-fructos-1-yl) amphotericin B - Oxonol V1 bis(3-propyl-5-oxoisoazol-4yl)pentamethine oxonol - SUV small unilamellar vesicles     

Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles.

6-Carboxyfluorescein was employed to examine the effect of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles. Proton permeability was measured by monitoring the decrease of 6-carboxyfluorescein fluorescence after a pH gradient from 3.5 (outside the vesicle) to 8.0 (inside the vesicle) was established. At 20 degrees C and below 1.2 M ethanol, the fluorescence decrease is best described by a single exponential function. Above 1.2 M ethanol, the intensity decrease is better described by a two-exponential decay law. Using the fitted rate constants and the vesicle radii determined from light-scattering measurements, the proton permeability coefficient, P, in DPPC vesicles was calculated as a function of ethanol concentration. At 20 degrees C, P increases monotonically with increasing ethanol content up to 1.0 M, followed by an abrupt increase at 1.2 M. The vesicle size also exhibits a sudden increase at around 1.2 M ethanol, which has been shown to result from vesicle aggregation rather than vesicle fusion. The abrupt increases in P and in vesicle size occur at the concentration region close to the critical ethanol concentration for the formation of the fully interdigitated gel state of DPPC. At 14 degrees C, the abrupt change in P shifts to 1.9-2.0 M ethanol, completely in accordance with the ethanol-temperature phase diagram of interdigitated DPPC. Effects of methanol and benzyl alcohol on lipid interdigitation have also been examined. At 20 degrees C, DPPC large unilamellar vesicles exhibit a dramatic change in P at 3 M methanol and at 40 mM benzyl alcohol. These concentrations come close to the critical methanol and benzyl alcohol concentrations for the formation of fully interdigitated DPPC structures determined previously by others. It can be concluded that proton permeability increases dramatically as DPPC is transformed from the noninterdigitated gel to the fully interdigitated gel state by high concentrations of alcohol. This marked increase in proton permeability can be attributed to the combined effect of the changes in membrane thickness and surface charge density, due to the ethanol-induced lipid interdigitation. The possible effects of the increased proton permeability caused by ingested ethanol on gastric mucosal membranes are discussed.     

Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls

We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K⁺/H⁺ antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K⁺ (with Cl⁻ or SCN⁻) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H⁺-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K⁺/H⁺ exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl⁻ and, second, efflux of K⁺ from K⁺-loaded vesicles drives intravesicular accumulation of H⁺ against the electrochemical potential gradient. Neither was the exchange due to competition between K⁺ and quinacrine for membrane sites, nor to inhibition of the H⁺-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K⁺ (at micromolar concentrations); Na⁺/H⁺ antiport was detected only at concentrations two orders of magnitude higher. Rb⁺, Li⁺, or Cs⁺ were ineffective. Dependence of tonoplast K⁺/H⁺ antiport on K⁺ concentration was complex, showing saturation at 10 millimolar K⁺ and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO₃⁻ and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to NO₃⁻ or oligomycin). The possible physiological role of such a K⁺/H⁺ exchange mechanism is discussed.     

Relationship between fluidity and ionic permeability of bilayers from natural mixtures of phospholipids

Summary Proton and calcium permeability coefficients of large unilamellar vesicles made from natural complex mixtures of phospholipids were measured in various conditions and related to membrane fluidity. Permeability coefficients at neutral pH and 25°C were in the range of 10⁴ cm sec¹ and 2.5×10¹¹ cm sec¹ for protons and calcium, respectively. With the exception of two cases. (H⁺)>10⁴ m and (Ca²⁺)>10³ m, fluidity increases correspond to permeability increases. Theoretical analysis shows that, for both ions, the measured values of permeability coefficients imply that the permeation process is controlled by the productD ₁ D ₂ of the diffusion coefficient from the medium into the membrane (D ₁) by the diffusion coefficient in the membrane (D ₂). Further analysis ofD ₁ values deduced from combined use of permeability and fluidity data shows that the solubilization should occur in a medium of dielectric constant of about 12, suggesting the involvement of the hydration water of membranes. High proton concentrations, although having virtually no effect on fluidity, trigger the appearance of (i) lateral heterogeneity in membranes, as seen by³¹P NMR, and (ii) large permeability increases. It is proposed that the main effect of fluidity and/or lateral heterogeneity on permeability may bevia the membrane hydration control. We conclude that the current assumption that permeability is controlled by fluidity should be regarded with caution, at least in the case of ions and natural mixtures of phospholipids.