Pump-Rest-Leak-Repeat: regulation of the mammalian-brain V-ATPase via ultra-slow mode-switching

Abstract

Vacuolar-type adenosine triphosphatases (V-ATPases) are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases. They hydrolyze ATP to establish electrochemical proton gradients for a plethora of cellular processes. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by ~1 V-ATPase molecule per synaptic vesicle. To shed light into this bona fide single-molecule biological process, we investigated electrogenic proton pumping by single mammalian-brain V-ATPases in single synaptic vesicles (SVs). We show V-ATPases do not pump continuously in time, as hypothesized by observing the rotation of bacterial homologs and assuming strict ATP/proton coupling. Instead, they stochastically switch between three ultra-long-lived proton-pumping, inactive, and proton-leaky modes. Surprisingly, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. Instead, ATP regulates V-ATPase activity via the switching probability of the proton-pumping mode. In 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/none stochastic fluctuations in the electrochemical gradient of SVs which 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 emphasises the mechanistic and biological importance of ultra-slow mode-switching.