Orthosteric and allosteric inhibition of the brain V-ATPase outside the alternating-access cycle

Active transporters move solutes uphill by harnessing the free energy stored in ATP or ion-motive gradients. A central tenet of molecular pharmacology is that orthosteric and allosteric inhibitors of active transporters act by slowing the transport cycle. We test this foundational premise, previously supported by ensemble-average data, at the single-molecule level. Using endogenous mammalian-brain V-ATPase, we show that bulk acidification can be abolished without any reduction in the single-molecule proton-transport rate. Instead, inhibitors predominantly promote reversible excursions outside the canonical transport cycle into ultralong-lived inactive modes. Overall, our findings establish the existence of two orthogonal axes of pharmacological control (on-cycle catalytic rates and off-cycle mode occupancy) that can be selectively or jointly actuated for precision drug design and likely generalize to other active transporters. V-ATPases are proton pumps that acidify synaptic vesicles and intracellular compartments. Here, authors show that several inhibitors mainly switch individual brain V-ATPases into long-lived inactive states instead of slowing proton pumping rates.