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

Kosmidis, E.; Fokaeos, M.; Shuttle, C. G.; Isselstein, M.; Cvjetkovic, A.; Johnson, P. J.; Pedersen, J. L.; Jahn, R.; Preobraschenski, J.; Stamou, D.

doi:10.1101/2025.09.28.679038

bioRxiv · 10.1101/2025.09.28.679038

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

Kosmidis, E.·Fokaeos, M.·Shuttle, C. G.·Isselstein, M.·Cvjetkovic, A.·Johnson, P. J.·Pedersen, J. L.·Jahn, R.·Preobraschenski, J.·Stamou, D.

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Abstract

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that establish proton gradients across cellular membranes1,2. Their pharmacological inhibition is currently under active investigation as a therapeutic strategy for cancer, infectious diseases, and autophagy-related disorders3,4. 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 rates5-7. 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 primary8,9 and secondary10-13 active transporters, our results suggest a novel strategy for therapeutic intervention that targets mode occupancy rather than the canonical transport cycle.

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Kosmidis, E., Fokaeos, M., Shuttle, C. G., Isselstein, M., Cvjetkovic, A., Johnson, P. J., Pedersen, J. L., Jahn, R., Preobraschenski, J., Stamou, D.. 2025-09-30. Pharmacological inhibition of V-ATPase targets mode-switching but not the proton transport cycle. https://doi.org/10.1101/2025.09.28.679038

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