TY - JOUR

T1 - Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching

AU - Kosmidis, Eleftherios

AU - Shuttle, Christopher G.

AU - Preobraschenski, Julia

AU - Ganzella, Marcelo

AU - Johnson, Peter J.

AU - Veshaguri, Salome

AU - Holmkvist, Jesper

AU - Møller, Mads P.

AU - Marantos, Orestis

AU - Marcoline, Frank

AU - Grabe, Michael

AU - Pedersen, Jesper L.

AU - Jahn, Reinhard

AU - Stamou, Dimitrios

N1 - Publisher Copyright: © 2022, The Author(s), under exclusive licence to Springer Nature Limited.

PY - 2022

Y1 - 2022

N2 - Vacuolar-type adenosine triphosphatases (V-ATPases)1–3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. 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 homologues8 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.

AB - Vacuolar-type adenosine triphosphatases (V-ATPases)1–3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. 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 homologues8 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.

U2 - 10.1038/s41586-022-05472-9

DO - 10.1038/s41586-022-05472-9

M3 - Journal article

C2 - 36418452

AN - SCOPUS:85142435227

VL - 611

SP - 827

EP - 834

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7937

ER -