Journal
NATURE
Volume 611, Issue 7937, Pages 827-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41586-022-05472-9
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Funding
- Novo Nordisk Foundation [NNF17OC0028176]
- Villum Foundation [17617, 17646]
- Lundbeck Foundation [R249-2017-1406, R250-2017-1175]
- European Research Council
- NIH [R01-AG057342]
- Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany's Excellence Strategy [EXC 2067/1-390729940]
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Vacuolar-type adenosine triphosphatases (V-ATPases) play a vital role in establishing electrochemical proton gradients in cells. This study reveals that V-ATPases do not pump continuously, but rather switch between different modes stochastically.
Vacuolar-type adenosine triphosphatases (V-ATPases)(1-3) are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases(4,5). They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes(1,3). In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle(6,7). To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues(8) and assuming strict ATP-proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching.
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