Pump, Rest and Repeat: Single Molecule Measurements Reveal Mode-Switching in the Mammalian Brain V-ATPase

Abstract

Vacuolar-type adenosine triphosphatases (V-ATPases) energize transport of neurotransmitters into neuronal secretory vesicles by increasing the lumenal pH via proton-pumping. Despite being ubiquitously found in neurons, their precise functional and regulatory characteristics remain elusive. Here, we performed a single molecule study on the rat brain V-ATPase in hybrid synaptic vesicles using fluorescence microscopy. The proton-pump stochastically switched modes between long-lived on-cycle, off-cycle and leaky states. Electrochemical gradients across the vesicle membrane regulate the lifetimes of these states as well as the pumping efficiency of the V-ATPase. The probability of the protein to be in either of these newly uncovered states is allosterically regulated by catalytic substrate concentration. Paradoxically, the pumping rates remain independent of ATP in physiologically relevant concentrations. These results reveal the crucial importance of mode-switching for the function and regulation of the V-ATPase.