Pharmacological inhibition of V-ATPase targets mode-switching but not the proton transport cycle

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that establish proton gradients across cellular membranes^1,2. Their pharmacological inhibition is currently under active investigation as a therapeutic strategy for cancer, infectious diseases, and autophagy-related disorders^3,4. However, the molecular mechanism underlying V-ATPase inhibition remains poorly understood. Based on ensemble average measurements, it is widely assumed that inhibitors suppress activity by slowing the catalytic transport cycle and reducing proton transport rates^5–7. Here, we tested this popular notion by directly measuring single-molecule proton pumping in the presence of three potent V-ATPase inhibitors: bafilomycin A1, concanamycin A, and diphyllin. Although all compounds abolish proton gradients in a canonical concentration-dependent manner (IC₅₀ of 0.2 nM, 0.6 nM, and 41 nM, respectively), they leave the proton transport rate of active V-ATPases essentially unchanged. Instead, inhibitors modulate the reversible switching kinetics between ultralong-lived active (pumping) and inactive modes. Distinct inhibitors modulate mode lifetimes in a mode-specific and differentially efficient manner, altering the probability of the pump being in the active mode. Given that mode-switching has been documented across diverse primary^8,9 and secondary^10–13 active transporters, our results suggest a novel strategy for therapeutic intervention that targets mode occupancy rather than the canonical transport cycle.