Voltage-dependent proton fluxes in liposomes

Liposomes containing buffered KCl were prepared from bacterial lipids, were diluted into K⁺-free media and were treated with valinomycin to induce the formation of a diffusion potential (Δψ). Upon formation of such a potential, substantial proton influx was observed, as assayed by the quenching of 9-aminoacridine fluorescence. Complete reversal of fluorescence quenching occurred when the potential was collapsed by addition of KCl or when methylamine was added. Studies of proton influx as a function of the theoretical magnitude of the Δψ indicated that the phenomenon occurred only above a Δψ of about -60 mV. Establishment of a Na⁺ diffusion potential also resulted in proton influx. Treatment of K⁺-loaded liposomes with N,N′-dicyclohexylcarbodiimide did not reduce the Δψ-dependent proton influx. Moreover, proton influx could be demonstrated upon imposition of a diffusion potential in liposomes prepared from a synthetic lipid. The proton fluxes associated with generation of a diffusion potential in liposomes may complicate studies of reconstituted systems in which proton translocation should occur, and may affect the magnitude of the electrochemical proton gradient that is operant under some conditions.