Role of cardiolipin in proton transmembrane flux and localization

In eukaryotic cells, the phospholipid cardiolipin (CL) is a crucial component that influences the function and organization of the mitochondrial inner membrane. In this study, we examined its potential role in passive proton transmembrane flux using unilamellar vesicles composed of natural egg phosphatidylcholine (PC) alone or with the inclusion of 18 or 34 mol % CL. A membrane potential was induced by a potassium gradient, and oxonol VI dye was used to monitor membrane potential dissipation resulting from proton transmembrane efflux. Increasing the CL content led to a net increase in proton efflux, which was also dependent on the magnitude of the membrane potential. The same increase in proton efflux was measured in the presence of the equally negatively charged phosphatidylglycerol, indicating that the charge of CL plays a more important role than its structure in this mechanism. When varying the proton membrane permeability (p_H) using the protonophore CCCP, we observed that unlike PC liposomes, where a small amount of CCCP was sufficient to achieve maximum flux, a significantly larger amount of protonophore was required in the presence of CL. Conversely, increasing the buffer capacity increased proton flux, indicating that proton availability, rather than membrane permeability, may be the limiting factor for proton leak. Our findings demonstrated that a higher proton content associated with the membrane was correlated with an increasing leak in the presence of CL. Additionally, smaller liposome diameters appeared to favor proton leak. Taken together, our results suggest that the presence of negatively charged CL in a membrane traps protons and increases their leakage, potentially in a manner dependent on membrane curvature. We discuss the possible mechanisms and implications of these findings for mitochondrial respiration function.