Determination of Proton Flux and Conductance at pH 6.8 through Single F^sub o^ Sectors from Escherichia coli

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single F^sub o^ of the F^sub 1^F^sub o^ ATP synthase. Lipid vesicles reconstituted with just a few functional F^sub o^ from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCI. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single F^sub o^ sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type F^sub o^ has a proton flux of 3100 ± 500 H+/s/F^sub o^ at a transmembrane potential of 106 mV (25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS. [PUBLICATION ABSTRACT]