Authors
Kosmidis E, Fokaeos M, Shuttle CG, Isselstein MC, Cvjetkovic A, Johnson PJ, Pederesen JL, Jahn R, Preobraschenski J, Stamou D
Journal
BioRxiv
Citation
bioRxiv 2025.09.28.679038.
Abstract
Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that establish proton gradients across cellular membranes. Their pharmacological inhibition is currently under active investigation as a therapeutic strategy for cancer, infectious diseases, and autophagy-related disorders. 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. 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 and secondary active transporters, our results suggest a novel strategy for therapeutic intervention that targets mode occupancy rather than the canonical transport cycle.
