Summary
We demonstrate that a simple kinetic model describes the transport of protons across lipid bilayer membranes by the weak acid CCCP (carbonyl cyanidem-chlorophenylhydrazone). Four parameters characterize this model: the adsorption coefficients of the anionic and neutral forms of the weak acid onto the interface (β A andβ HA) and the rate constants for the movement of A− and HA across the membrane (k A andk HA). These parameters were determined by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. From these equilibrium and steady state measurements on diphytanoyl phosphatidylcholine/chlorodecane membranes we found thatβ A=β HA=1.4 10-3cm,k A=175 s−1 andk HA=12,000 sec−1. These parameters and our model describe our kinetic experiments if we assume that the protonation reactions, which occur at the interfaces, remain at equilibrium. The model predicts a single exponential decay of the current in a voltage-clamp experimetn. The model also predicts that the decay in the voltage across the membrane following an intense current pulse of short duration (≈50 nsec) can be described by the sum of two exponentials. The magnitudes and time constants of the relaxations that we observed in both voltage-clamp and charge-pulse experiments agree well with the predictions of the model for all values of pH, voltage and [CCCP].
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References
Anderson, O., Feldberg, S., Nakadomari, H., Levy, S., McLaughlin, S. 1978. Electrostatic potentials associated with the absorption of tetraphenylborate into lipid bilayer membranes.In: Membrane Transport Processes. Vol. 2, pp. 325–334. D.C. Tosteson, Yu.A. Ovchinnikov, and R. LaTorre, editors. Raven Press, New York
Aveyard, R., Haydon, D.A. 1973. An Introduction to the Principles of Surface Chemistry. Cambridge University Press, London
Bangham, A.D., Hill, M.W., Miller, N.G.A. 1974. Preparation and use of liposomes as models of biological membranes.Methods Membr. Biol. 1:1–68
Barenholz, Y., Gibbes, D., Litman, B.J., Goll, J., Thompson, T.E., Carlson, F.D. 1977. A simple method for the preparation of homogeneous phospholipid vesicles.Biochemistry 16:2806–2810
Benz, R., Läuger, P., Janko, K. 1976. Transport kinetics of hydrophobic ions in lipid bilayer membranes: Charge pulse relaxation studies.Biochim. Biophys. Acta 455:701–720
Benz, R., McLaughlin, S. 1983. The molecular mechanism of action of the proton ionophore FCCP (carbonylcyanidep-trifluoromethoxyphenylhydrazone).Biophys. J. 41:381–398
Bhattacharyya, P., Barnes, E.M., Jr. 1976. ATP-dependent calcium transport in isolated membrane vesicles fromAzobacter vinelandii.J. Biol. Chem. 251:5614–5619
Cohen, F.S., Eisenberg, M., McLaughlin, S. 1977. The kinetic mechanism of action of an uncoupler of oxidative phosphorylation.J. Membrane Biol. 37:361–396
Dilger, J., McLaughlin, S. 1979. Proton transport through membranes induced by weak acids: A study of two substituted benzimidazoles.J. Membrane Biol. 46:359–384
Eigen, M., Kruse, W., Maass, G., De Maeyer, L. 1964. Rate constants of protolytic reactions in aqueous solution.Prog. React. Kinet. 2:287–318
Grinius, L.L., Jasaitis, A.A., Kadziauskas, Yu.P., Liberman, E.A., Skulachev, V.P., Topali, V.P., Tsofina, L.M. Vladimirova, M.A. 1970. Conversion of biomembrane-produced energy into electric form: I. Submitochondrial particles.Biochim. Biophys. Acta 216:1–12
Hall, J.E., Mead, C.A., Szabo, G. 1973. A barrier model for current flow in lipid bilayer membranes.J. Membrane Biol. 11:75–97
Harold, F.M. 1977. Membranes and energy transduction in bacteria.Curr. Top. Bioenerg. 6:83–149
Heytler, P.G. 1963. Uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones: I. Some characteristics ofm-Cl-CCP action on mitochondria and chloroplasts.Biochemistry 2:357–361
Heytler, P.G., Prichard, W.W. 1962. A new class of uncoupling agents—carbonyl cyanide phenylhydrazones.Biochem. Biophys. Res. Commun. 7:272–275
Hladky, S.B. 1974. The energy barriers to ion transport by nonaction across thin lipid membranes.Biochim. Biophys. Acta 352:71–85
Hopfer, U., Lehninger, A.L., Thompson, T.E. 1968. Protonic conductance across phospholipid bilayer membranes induced by uncoupling agents for oxidative phosphorylation.Proc. Natl. Acad. Sci. USA 59:484–490
LeBlanc, O.H., Jr. 1971. The effect of uncouplers of oxidative phosphorylation on lipid bilayer membranes: Carbonylcyanidem-chlorophenylhydrazone.J. Membrane Biol. 4:227–251
Lee, A.G. 1978. Effects of charged drugs on the phase transition temperatures of phospholipid bilayers.Biochim. Biophys. Acta 514:95–104
Liberman, E.A., Topaly, V.P., Tsofina, L.M., Jasaitis, A.A., Skulachev, V.P. 1969. Mechanism of coupling of oxidative phosphorylation and the membrane potential of mitochondria.Nature (London) 222:1076–1078
Lowry, R.R., Tinsley, I.J. 1974. A simple sensitive method for lipid phosphorus.Lipids 9:491–492
McLaughlin, A., Eng, W.-K., Vaio, G., Wilson, T., McLaughlin, S. 1983. Dimethonium, a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes.J. Membrane Biol. 76:183–193
McLaughlin, S. 1977. Electrostatic potentials at membranesolution interfaces.Curr. Top. Membr. Transp. 9:71–144
McLaughlin, S.G.A., Dilger, J.P. 1980. Transport of protons across membranes by weak acids.Physiol. Rev. 60:825–863
Mitchell, P. 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism.Nature (London) 191:144–148
O'Shaughnessy, K., Hladky, S.B. 1983. Transient currents carried by the uncoupler, carbonyl cyanidem-chlorophenylhydrazone.Biochim. Biophys. Acta 724:381–387
Peterkofsky, A., Gazdar, C. 1979.Escherichia coli adenylate cyclase complex: Regulation by the proton electrochemical gradient.Proc. Natl. Acad. Sci. USA 76:1099–1103
Ramos, S., Schuldiner, S., Kaback, H.R. 1976. The electrochemical gradient of protons and its relationship to active transport inEscherichia coli membrane vesicles.Proc. Natl. Acad. Sci. USA 73:1892–1896
Reyes, J., Benos, D.J. 1984. Changes in interfacial potentials induced by carbonylcyanide-phenylhydrazone uncouplers Possible role in inhibition of mitochondrial oxygen consumption and other transport processes.Membr. Biochem. 5:243–268
Sjogren, R.E., Gibson, M.J. 1981. Bacterial survival in a dilute environment.Appl. Environ. Microbiol. 41:1331–1336
Skulachev, V.P., Sharaf, A.A., Liberman, E.A. 1967. Proton conductors in the respiratory chain and artificial membranes.Nature (London) 216:718–719
Tanford, C. 1978. The hydrophobic effect and the organization of living matter.Science 200:1012–1018
Terada, H. 1981. The interaction of highly active uncouplers with mitochondria.Biochim. Biophys. Acta 639:225–242
Tsien, R.Y., Hladky, S.B. 1982. Ion repulsion within membranes.Biophys. J. 39:49–56
Verkman, A.S., Solomon, A.K. 1980. Kinetics of phloretin binding to phosphatidylcholine vesicle membranes.J. Gen. Physiol. 75:673–692
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Kasianowicz, J., Benz, R. & McLaughlin, S. The kinetic mechanism by which CCCP (carbonyl cyanidem-Chlorophenylhydrazone) transports protons across membranes. J. Membrain Biol. 82, 179–190 (1984). https://doi.org/10.1007/BF01868942
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DOI: https://doi.org/10.1007/BF01868942