valinomycin and pyranine

ortho-Carborane (1,2-C2B10H12) was found to be a carrier of protons in both mitochondrial and artificial lipid membranes, suggesting that this dicarborane can reversibly release hydrogen ions and diffuse through the membranes in neutral and anionic forms. Similar to conventional uncouplers (e.g. 2,4-dinitrophenol), o-carborane stimulated mitochondrial respiration and decreased the membrane potential at concentrations of tens of micromoles. Protonophoric activity of o-carborane was observed both by a fluorometric assay using pyranine-loaded liposomes and electrical current measurements across planar lipid bilayers. Substantial contribution of the proton flux to the o-carborane-mediated current was proved by a shift of the zero current voltage upon imposing a pH gradient across the membrane. Meta-carborane (1,7-C2B10H12) lacked the protonophoric activity in line with its reduced C-H acidity. The results suggest that weak C-H acids can exhibit protonophoric activity in the biological environment. The finding of a new class of protonophoric compounds is of substantial interest due to promising anti-obesity and anti-diabetic properties of uncouplers.

Topics: Animals; Arylsulfonates; Boranes; Boron Compounds; Hydrogen-Ion Concentration; Kinetics; Lewis Acids; Lipid Bilayers; Liposomes; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial Membranes; Phosphatidylcholines; Rats; Uncoupling Agents; Valinomycin

There is an acute need for a functional assay allowing the investigation of efflux pumps. A dedicated procedure was previously developed, but although it was unambiguous, it suffered from a lack of reproducibility. We describe an optimization of the procedure that makes the assay much more robust.

Topics: Arylsulfonates; Biological Assay; Biological Transport; Hydrogen-Ion Concentration; Liposomes; Proteolipids; Valinomycin

Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into preformed asolectin liposomes which contained 120 mM KCl as internal high K+ medium. 45Ca2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H+i/45Ca2+o or Na+i/45Ca2+o exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca2+ uptake in liposomes was studied in parallel. Na+i/45Ca2+o exchange activity was not detectable. H+i/45Ca2+o exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100-200 nmoles 45Ca2+ per mg protein in 30 s. 45Ca2+ uptake in voltage-dependent assays (a K+ diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca2+/H+ countertransport. H+i/45Ca2+o uptake showed a saturable component at around 80 microM Ca and was coupled to an increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K+ medium. The results are consistent with the presence of a functional nH+/Ca2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H+/45Ca2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H+/Ca2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.

Topics: Animals; Antiporters; Arylsulfonates; Calcium; Calcium Radioisotopes; Calcium-Binding Proteins; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carrier Proteins; Cation Transport Proteins; Chromatography, Affinity; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Proteins; Microscopy, Electron; Mitochondria, Liver; Octoxynol; Phosphatidylcholines; Phospholipids; Rats; Valinomycin

1. The effects of alkaline-inside pH gradients on 36Cl- uptake were quantified by using brush-border membrane (BBM) and basolateral membrane (BLM) vesicles from guinea-pig jejunum. 2. With BBM vesicles, a pHo/pHi gradient of 5.0/7.5 yielded fast overshoots involving a random, non-obligatory Cl(-)-H+ symport, strongly inhibited by CCCP. In contrast, BLM vesicles responded to similar pH gradients with much smaller, delayed overshoots, unaffected by CCCP. 3. The initial Cl- entry rates into BLM vesicles were a function of each pHo, pHi and delta pH value. They were stimulated by valinomycin in the presence of inward-directed K+ gradients. Short-circuiting the membrane potential with equilibrated K+ and valinomycin inhibited pH gradient-dependent Cl- uptake, but only partially. 4. Taken together, these results indicate that guinea-pig jejunal BLM vesicles possess both Cl- conductance and Cl(-)-H+ symport activities. 5. Even when different, the BBM and the BLM symporters are mechanistically similar. Neither of them involves a Cl(-)-OH- antiport, nor a simultaneous Cl(-)-anion exchange mechanism. Rather, for each membrane, all of these activities (symport, anion exchange) can be explained in terms of a single mobile carrier acting as a random, non-obligatory Cl(-)-H+ symporter where exchange occurs simply by counterflow. Net Cl- translocation via either the ternary (Cl(-)-C-H+) or the binary (Cl(-)-C) complexes accounts, respectively, for the existence of two, operationally distinct, electroneutral and rheogenic components. 6. The BBM symporter appears to involve an AE2 protein, but the molecular identity of the BLM one remains to be established.

Topics: Animals; Arylsulfonates; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Chloride Channels; Chlorides; Fluorescent Dyes; Guinea Pigs; Hydrogen-Ion Concentration; Ionophores; Jejunum; Kinetics; Membrane Potentials; Microvilli; Potassium; Protons; Time Factors; Valinomycin

The decay of the pH difference (delta pH) across soyabean phospholipid vesicular membrane (created by temperature jump), by the combined action of valinomycin and 2,4-dinitrophenol (DNP) has been monitored with the help of fluorescence from pyranine entrapped inside the vesicles under a variety of concentration conditions. The results suggest the following for the pH region of our interest (pH approximately 6 to pH approximately 8): (i) The rate limiting step in the proton transport cycle is not the transport of proton as DNPH, but the back transport of DNP- and the alkali metal ion M+ as Val-M(+)-DNP- across the membrane. The rate constant associated with the transport of the ternary complex has been estimated to be approximately 1.5 x 10(3) s-1. (ii) The dissociation constant of the ternary complex Val-M(+)-DNP- in the membrane are approximately 1 mM for M+ = K+ and approximately 0.001 mM for M+ = Na+. (iii) The reduction in the cation selectivity of valinomycin on complexing with DNP- is much more than that observed with the anionic form of carbonyl cyanide m-chlorophenylhydrazone (CCCP). The results also provide a verification of a corollary of Mitchell's hypothesis: an experimental strategy which enhances the delta pH decay rate should also be a strategy for the efficient uncoupling of oxidative and photophosphorylation.

Topics: 2,4-Dinitrophenol; Arylsulfonates; Biological Transport; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Dinitrophenols; Fluorescent Dyes; Hydrogen; Hydrogen-Ion Concentration; Kinetics; Liposomes; Phospholipids; Potassium; Sodium; Uncoupling Agents; Valinomycin

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.

Topics: Arylsulfonates; Benzofurans; Cations; Ethers, Cyclic; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Mathematics; Membrane Potentials; Permeability; Potassium Chloride; Protons; Valinomycin

Topics: Arylsulfonates; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Mitochondria; Models, Biological; Oleic Acid; Oleic Acids; Valinomycin

Pyranine was incorporated into sonicated unilamellar vesicles of soybean phosphatidylcholine to monitor changes in the internal pH of the vesicles. Dilution of soybean phosphatidylcholine vesicles loaded with 0.3 M KCl, KNO3 or K2SO4 into salt-free buffer resulted in rapid exchange of K+ and protons. A pseudoequilibrium distribution of ions was achieved, since addition of valinomycin, uncoupler or nigericin now caused a rapid alkalinization of the vesicle interior. Dilution into buffer containing NaCl gave a further exchange of Na+ and protons following the initial K+/proton exchange. Na+ permeation was slower than that of K+. A stable membrane potential was not generated by the ion movements. It is proposed that aqueous channels are formed through the phospholipid bilayers and that K+ and Na+ permeate through these channels as the hydrated ions.

Topics: Arylsulfonates; Cations; Coloring Agents; Glycine max; Hydrogen-Ion Concentration; Ion Channels; Liposomes; Membrane Potentials; Nigericin; Nitrates; Particle Size; Phosphatidylcholines; Potassium; Potassium Chloride; Potassium Compounds; Protons; Salicylanilides; Sodium; Sulfates; Valinomycin

The fluorescent indicator pyranine was used for recording the internal pH of liposomes. The proton permeability was deduced from the velocity of the internal pH increase which was caused by shifting the external pH from 7 to 9. From valinomycin titration of the proton permeability in the presence of internal and external KCl (0.1 M), the permeability coefficient of H+ (PH) was obtained as 10(-4) cm/s at 22 degrees C. The coefficient was twice this value with the ATP synthase isolated from Wolinella succinogenes present in the liposomal membrane (10 mg protein/g phospholipid). ADP and phosphate had no effect on the latter PH. The protonophore TTFB (5 mumol/g phospholipid) increased the PH by 3 orders of magnitude. The permeability coefficients of H+ and K+ were used for calculating the delta uH and the proton flux associated with the phosphorylation which was driven by gradients of H+ and K+. For the conditions of limiting permeability of K+, the following conclusions were drawn. (1) In the steady state of rapid ion flux, the electrical potential across the liposomal membrane as calculated according to the Goldman equation, is directed opposite to the corresponding Nernst potential which is calculated from the K+ gradient. (2) The maximum turnover numbers of phosphorylation require a delta uH of 200-220 mV across the liposomal membrane. These values of delta uH and the corresponding turnover numbers are close to those brought about by the bacterial electron transport and the coupled phosphorylation. (3) The velocity of phosphorylation is linearly related to the proton flux. The slope of the line can be explained on the basis of an H+/ATP ratio of approx. 3.

Topics: Arylsulfonates; Bacteroidaceae; Benzimidazoles; Cell Membrane Permeability; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Phosphorylation; Potassium; Proton-Translocating ATPases; Protons; Spectrometry, Fluorescence; Uncoupling Agents; Valinomycin

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.

Topics: Arylsulfonates; Buffers; Diffusion; Hydrogen-Ion Concentration; Hydroxides; Kinetics; Liposomes; Mathematics; Membrane Potentials; Models, Biological; Protons; Solutions; Thermodynamics; Valinomycin

The effects of small hydrophobic molecules on valinomycin-mediated K+ transport in small unilamellar soybean phospholipid vesicles have been studied by using a vesicle-entrapped pH-sensitive hydrophilic fluorescence probe to monitor counterion-limited, passive H+ diffusion into vesicles after an abrupt decrease in external pH [Clement, N. R., & Gould, J. M. (1981) Biochemistry (preceding paper in this issue)]. Under conditions where, even in the absence of valinomycin, transmembrane KL+ movement represented the primary and limiting counterion flux, less than 1 valinomycin molecule/vesicle was sufficient to accelerate the rate of H+ entry into all of the vesicles. Incorporation of the bulkily substituted molecules butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and p-di-tert-butylbenzene into soybean lipid bilayers had no effect upon K+ diffusion in the absence of valinomycin. However, the presence of these hydrophobic molecules increased the apparent efficacy for K+ transport of a given valinomycin concentration by as much as 4-6 fold. The less bulky membrane perturbants tert-butyl alcohol, phenol, and heptane showed very much less dramatic effects. While the rate of valinomycin-mediated K+ transport (in the presence or absence of BHT) was very sensitive to temperature-induced changes in membrane fluidity, the degree of synergistic interaction between valinomycin and BHT was independent of temperature. Furthermore, BHT, BHA, and p-di-tert-butylbenzene, at levels which alter valinomycin-mediated K+ transport, did not by themselves induce changes in membrane fluidity. It is postulated that changes in phospholipid head-group packing and/or surface charge density brought about by the presence of bulky perturber molecules leads to changes in partitioning of valinomycin or the valinomycin-K+ complex between the aqueous and membrane phases.

Topics: Arylsulfonates; Benzene Derivatives; Biological Transport; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Dose-Response Relationship, Drug; Drug Synergism; Fluorescent Dyes; Hydrogen-Ion Concentration; Liposomes; Potassium; Pyrenes; Valinomycin