Unilamellar liposomes were prepared by extrusion through 0.1-μm-pore size Nucleopore polycarbonate membranes using an Avanti Mini-Extruder. The suspension was vortexed and then freeze-thawed three times. The lipid was then resuspended in buffer (100 mM KCl, 20 mM MES, 20 mM MOPS, 20 mM Tricine titrated with KOH to pH 6.0) containing 0.5 mM pyranine. To prepare pyranine-loaded liposomes, lipid (2 mg POPC, 1 mg POPG and 1 mg cholesterol) in a chloroform suspension was dried in a round-bottom flask under a stream of nitrogen. The lumenal pH of the liposomes was assayed with pyranine by a slightly modified procedure of.
Detection of proton transport in pyranine-loaded liposomes Here, we report synthesis of a mitochondria-targeted derivative of CCCP (a conjugate with TPP coined mitoCCCP), which appeared to be a rather weak uncoupler but exhibited effective inhibition of the CCCP-caused uncoupling in mitochondria.Ģ.10. However, the resulting compound lacked the uncoupling activity. It was assumed that the addition of a lipophilic cation will provide selective accumulation of the uncoupler in the mitochondria, since energized mitochondria are negatively charged with respect to the cytosol. Earlier in the laboratory of Michael Murphy, an attempt was made to obtain a mitochondria-targeted uncoupler based on a conjugate of the popular uncoupler DNP with a triphenylphosphonium (TPP) cation.
to avoid adverse effects, it is reasonable to design mitochondria-targeted agents. To make the impact of uncouplers more selective, i.e. By contrast, kCh was ineffective with 2,4-dinitrophenol (DNP), fatty acids and gramicidin A. Later, the kCh-induced recoupling was also reported for curcumin derivatives, eudesman, SR4, a bicyclic hydroquinone and BAM15. an increase in the membrane dipole potential and diminution of membrane fluidity. Nevertheless, it cannot be excluded that the recoupling action of kCh is somehow associated with its impact on physical properties of membranes, i.e. This idea was supported by the fact that in planar bilayer lipid membranes, kCh not only failed to reverse the protonophoric action of SF6847, but even enhanced the conductivity increase caused by this uncoupler. In particular, the recoupling effect of 6-ketocholestanol (kCh) found earlier by Starkov and colleagues was tentatively considered as an evidence in favor of involvement of proteins in mitochondrial uncoupling mediated by such agents as carbonyl cyanide- m-chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and tyrphostin A9 (SF6847). There is so far no consensus whether the uncoupling action occurs with or without the aid of membrane proteins, despite the general confidence in the protonophoric nature of this action. Although the experiments with uncouplers on artificial membranes, mitochondria and bacteria have played a crucial role in the validation of Mitchell’s chemiosmotic theory, the mechanism of uncoupling remains not fully understood. Compounds that compromise the coupling between respiration and phosphorylation called uncouplers have recently received strong interest as promising anti-cancer, antibacterial, anti-obesity, antidiabetic, neuroprotective and cardioprotective agents. It is now generally accepted that electron transfer via a chain of proton pumps in the inner mitochondrial or bacterial membrane results in the formation of a transmembrane difference of electrochemical potentials of hydrogen ions that couples the oxidation of respiratory substrates to ATP synthesis.