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.

Grabe for providing the MATLAB code for fitting the non-equilibrium physical model. We thank John Rubinstein for kindly providing us with the purified bacterial effector protein SidK. Huber for inspiring discussions that led to the inception of this project.

We thank Helmut Grubmueller and Carsten Kutzner for kindly providing us with the atomic model of the synaptic vesicle. This work was supported by the Lundbeck Foundation (Professorship grant R441-2023-360) and the Novo Nordisk Foundation (NNF17OC0028176). R.J. was supported by an ERC Advanced Grant (SVNeuroTrans).

This work was also supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) through grants SFB1690/A03 and Cluster of Excellence (EXC2067) Multiscale Bioimaging EXC 2067/1-390729940 (JP). Present address: Cancelight, Munich, Germany Present address: Sahlgrenska Center for Cancer Research, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden These authors contributed equally: Eleftherios Kosmidis, Maria Fokaeos. Department of Chemistry, University of Copenhagen, Copenhagen, Denmark Eleftherios Kosmidis, Maria Fokaeos, Christopher G.

Shuttle, Michael Isselstein, Alessandra Narducci, Aleksander Cvjetkovic & Dimitrios Stamou Eleftherios Kosmidis, Maria Fokaeos, Christopher G. Shuttle & Dimitrios Stamou Department of Mathematics, University of Manchester, Manchester, United Kingdom Department of Mathematics, University of Copenhagen, Copenhagen, Denmark Laboratory of Neurobiology, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany Center of Membrane Biochemistry and Lipid Research, Dresden University of Technology, Dresden, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 1690(CRC1690), University of Göttingen, Göttingen, Germany Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), Georg-August-University Göttingen, Göttingen, Germany D.S. is the founder of Illum Biotech. The other authors declare no competing interests.