A key enzyme in brain function shuts off randomly, study reveals

A new study conducted by researchers at the University of Copenhagen has found that V-ATPase, an enzyme thought to be a key component of brain function, switches off randomly, even for hours at a time. This discovery has the potential to change our understanding of how our brain functions, according to a press release.

V-ATPase is an enzyme that can break down ATP molecules, the cell's energy currency, as they pump protons across cellular membranes. The enzyme is found in all cell types of mammals; however, it has been attributed to an important role in cells of the brain where its action loads neurotransmitters into synaptic vesicles.

Simply put, the enzyme is responsible for providing energy to fill up the membrane bladders between neighboring neurons with chemicals that are needed to transfer a message between them. Therefore, the enzyme is quite crucial for neuronal communication, or that's what researchers have thought so far.

Working with rat neurons for the first time

Previous research about V-ATPase and its activity has been conducted using enzymes that were sourced from bacteria. However, for their research, the team at the University of Copenhagen decided to use V-ATPase, which was sourced from rat brains.

The researchers found that the membrane bladder of each neuron contained exactly one V-ATPase enzyme molecule. Furthermore, the enzyme appears to switch off at random intervals, putting an abrupt stop to the neurotransmitter-loading process. Overall, the enzyme is inactive about 40 percent of the time.

This has stumped the researchers since the finding shatters our understanding of how neurons work. Without an active enzyme, the membrane bladders or synapse should ideally be empty and incapable of transmitting signals, or the neurons have an alternate way of conveying information that scientists do not know about.

Going beyond neuroscience

As mentioned earlier, V-ATPase is present in all mammalian cell types and has piqued scientific curiosity for its role in cancer metastasis and other life-threatening diseases. Therefore, V-ATPase is a potential drug target that is being explored by the scientific community at large.

When screening drugs, researchers tend to simultaneously use signals from billions of enzyme molecules. Since enzymes are presumed to work constantly, knowing the average effect of the drug is sufficient for researchers in most cases.

In the case of V-ATPase though, we now know that the enzyme can shut off for long periods of time and jump back to activity. To know whether a drug really works well, the researchers need to be able to measure the behavior down to one molecule to determine if the drug will deliver its desired effect.

The research done by the Copenhagen team now lets researchers measure this behavior and can detect currents that are as much as a million times smaller than conventional methods, the press release said.

The research findings were published in the journal Nature.

Abstract

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