Protonic conductance across phospholipid bilayer membranes induced by uncoupling agents for oxidative phosphorylation.

The chemiosmotic coupling hypothesis for oxidative phosphorylation proposed by Mitchell1 postulates that electron transport along the respiratory chain in the mitochondrial membrane generates a transmembrane pH gradient, which becomes the immediate driving force for the enzymatic synthesis of adenosine 5'-triphosphate (ATP) from adenosine 5'-diphosphate (ADP) and phosphate through the reverse action of a vectorial ATPase system in the membrane. Mitchell has proposed that uncoupling agents such as 2,4-dinitrophenol, which uncouple phosphorylation of ADP from electron transport, are not inhibitors of specific enzymatic reactions per se, but act as lipid-soluble proton donor-acceptor systems that dissolve in the lipid phase of the membrane and conduct protons across it, thus preventing formation of pH gradients during electron transport. Experimental evidence for and against the Mitchell hypothesis has been discussed. 2-4 The availability of phospholipid bilayer systems formed in apertures separating two aqueous phases5-7 has provided an opportunity to determine whether these simple model systems, which are devoid of proteins or enzymes, respond to uncoupling agents in the manner postulated by Mitchell. The thickness, high electrical resistance and capacitance, and high-water permeability of these membranes closely resemble those of natural membranes.8 In an earlier communication from this laboratory,9 it was shown that 2,4-dinitrophenol greatly increased the electrical conductance of such phospholipid bilayer membranes. The increase was sufficient to account for the uncoupling action of dinitrophenol in mitochondria, on the assumption that the chemi-osmotic coupling hypothesis is correct. However, the data did not allow a conclusion as to whether 2,4dinitrophenol caused a specific protonic conductance or merely caused a generalized increase in permeability of the bilayer membrane to all cations (and anions) in the system studied. In this communication data are presented showing that in addition to 2,4dinitrophenol, the carbonylcyanide phenylhydrazone uncoupling agents also produce large increases in the specific conductance of phospholipid bilayer membranes, which are sufficient to account for their uncoupling activity. Each uncoupling agent was found to have a characteristic pH at which it is maximally effective. The increase in electrical conductance was found to be almost entirely the result of transport of protons across the membrane. Experimetal Methods.-The phospholipid bilayers were formed as described by Huang et al."'-12 The bulk lipid solutions employed consisted of 0.5-1.0% purified egg phosphatidyl choline and 0.25-0.50% cholesterol in n-decane. In some experiments 0.05-0.1% tocopheryl succinate was added. The aqueous phases consisted of 0.1 M