Proton channels are essential for many of the processes of life. The influenza A viral protein M2 is responsible for sensing the conditions necessary for viral RNA release. The proton-translocating FoF1 ATPase (ATP synthase) uses a proton gradient to drive adenosine triphosphate (ATP) synthesis. We have directly measured proton uptake in vesicles containing reconstituted M2 or FO by monitoring external pH after addition of valinomycin to vesicles with 100-fold diluted external [K+]. This proton flux assay was utilized to quantify proton flux through single M2 and Fo channels. Contrary to previous reports, proton uptake by M2 was not significantly altered by acidification of the extravesicular pH. We conclude that pH only weakly affects proton flux through M2 in the pH range of 5.4 - 7.0. Theoretical analysis utilized for such vesicle uptake assays illuminates the appropriate time scale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 seconds after ionophore addition yielded time-averaged single channel conductances of 0.35±0.2 aS and 0.72±0.4 aS at pH 5.4 and 7.0 respectively. Such a low time-average conductance implies that M2 is only conductive 10^-6 to 10^-4 of the time. M2 selectivity for hydrogen over potassium is ~10^7. Fo translocates protons across membranes, converting electrochemical energy to rotational inertia. Previous experiments have been partially confounded by a contaminating channel, CL, which co-purifies with Fo and leaks cations. CL activity is shown to not decrease following deletion of the previously uncharacterized yraM open reading frame of E. coli. Fo purified from a deletion strain lacking yraM is just as active as Fo purified from the wild-type strain. Using Fo from the deletion strain, the single-hit hypothesis of DCCD inhibition of passive proton flux through Fo was examined. A DCCD-induced reduction in ATP synthase activity correlates with a reduction in the total initial slope, the number of functional Fo per µg protein, and the single channel proton flux. At least 2 DCCD per Fo are required to totally inactivate passive proton flux. M2 and Fo have similar single channel conductances but different open probabilities.

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