Abstract
Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H+ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C12TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C12TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C12TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.
Highlights
The concept of ‘‘mild uncoupling’’ implies favorable therapeutic action of low concentrations of uncouplers which slightly decrease membrane potential thereby preventing fast production of reactive oxygen species (ROS) in mitochondria [1,2,3]
To study the effect of the mitochondria-targeted penetrating cations on the uncoupler-mediated proton flux through the planar bilayer lipid membrane (BLM), we measured proton diffusion potential, i.e. an electrical potential difference (DY) across the bilayer lipid membranes (BLM), generated as a result of transmembrane H+ flux facilitated by a protonophore under the conditions of pH difference between two BLM-separated compartments [31,32,33]
Summarizing the above presented data, one can conclude that C12-TPP, SkQ1 and other hydrophobic penetrating cations are able to promote the protonophorous action of anionic uncouplers on model lipid membranes (Figs.2, 3) and certain natural membranes (Figs.6, 7, 8, and 9)
Summary
The concept of ‘‘mild uncoupling’’ implies favorable therapeutic action of low concentrations of uncouplers which slightly decrease membrane potential thereby preventing fast production of reactive oxygen species (ROS) in mitochondria [1,2,3]. It has been assumed that toxic action of DNP and its analogues is related to an excessive decrease in mitochondrial membrane potential. Comparison of toxic doses for mice with uncoupling concentrations for isolated mitochondria has revealed poor correlation of these parameters for a large set of DNP analogues as well as for some other uncouplers [13]. These findings suggest that the toxic effect of at least some uncouplers in vivo could be unrelated to their action on mitochondria. It seems possible that the therapeutic window for uncouplers could be enlarged by agents promoting the uncoupling action of their small concentrations
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