Properties and new methods of non-equilibrium membrane bound proton fraction research under conditions of proton pump activation

Under the conditions of low-amplitude mitochondrial swelling, the oxidative phosphorylation system functions in a local coupling mode postulated by Williams in 1961. The proton pumps activation leads to the formation of non-equilibrium membrane bounded proton fraction (nef-H⁺), which is sorbed on the outer side of the inner mitochondrial membrane under these conditions. This proton fraction is crucial for the ATP synthesis. The present work is devoted to the development of the methods allowing investigations of the properties of nef-H⁺. For this purpose, a new membranotropic highly hydrophobic uncoupler 2,4,6-trichloro-3-pentadecylphenol was synthesized. In accordance with our results, it can be referred to as a new type of transmemebrane proton carriers, which interact specifically only with nef-H⁺ outer side of the inner membrane. A new method of the nef-H⁺ removal from the outer side of the inner mitochondrial membrane under the conditions when the proton pumps are active has been developed. The method is based on neutralization of nef-H⁺ by hydroxyl anions transferred by H₂PO₄⁻/OH⁻-antiporter to interfacial border. It was demonstrated that the membrane proton fraction is heterogeneous. Two components of the fraction were identified. And at the same time, it was shown that nucleotide translocator participates in the formation of one component. Also, in this series of experiments it was found that a well-known effect of respiration inhibition by high concentrations of uncoupler under certain conditions can be completely explained by the nef-H⁺ formation. We presume that one of the most important results of the studies carried out is a new independent evidence of the existence of nef-H⁺, which is formed under the conditions of proton pump activation.