Mechanism of the uncoupling of oxidative phosphorylation by gramicidin

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Abstract
The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of .DELTA..cxa..mu.H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and .DELTA.pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential. We conclude that decoupling of oxidative phosphorylation by gramicidin does not depend on transmembrane cation transport but results from specific interference with direct proton transfer to the H+-ATPase.