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

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, using individual synaptic vesicles fused with immobilized liposomes. 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 novel ultra-long-lived proton-pumping, inactive, and proton-leaky modes. Upending conventional wisdom, 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. This work reveals and emphasises the mechanistic and biological importance of mode-switching in protein regulation

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

Vacuolar-type adenosine triphosphatases (V-ATPases) are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases. They hydrolyse 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 about one V-ATPase molecule per synaptic vesicle. 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 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

Direct observation of proton pumping by a eukaryotic P-type ATPase

In eukaryotes, P-type ATPases generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements combined with a physical non-equilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 s) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states, but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates, but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters