Vacuolar-type adenosine triphosphatases (V-ATPases)^1,2,3 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 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.

液泡型腺苷三磷酸酶 (V-ATPases) ^1,2,3是产电旋转机械酶，在结构上与 F 型 ATP 合酶^4,5相关。它们水解 ATP 为大量细胞过程建立电化学质子梯度^1,3。在神经元中，所有神经递质加载到突触小泡中是由每个突触小泡约一个 V-ATPase 分子激活的^6,7。为了阐明这种真正的单分子生物学过程，我们研究了单个哺乳动物脑 V-ATP 酶在单个突触小泡中的电生质子泵送。在这里，我们表明 V-ATP 酶不会及时连续泵送，正如通过观察细菌同系物^{8的旋转所建议的那样}并假设严格的 ATP-质子耦合。相反，它们随机地在三种超长寿命模式之间切换：质子泵送、不活跃和质子泄漏。值得注意的是，对泵送的直接观察表明，生理相关的 ATP 浓度不会调节内在泵送速率。ATP 通过质子泵模式的切换概率调节 V-ATPase 活性。相比之下，电化学质子梯度调节泵送速率以及泵送和非活动模式的切换。模式切换的直接后果是突触小泡电化学梯度的全或无随机波动，预计会在质子驱动的神经递质二次活性加载中引入随机性，因此可能对神经传递具有重要意义。