A major discovery about mammalian brains surprises researchers

Summary: V-ATPase, a vital enzyme that enables neurotransmission, can be switched on and off randomly, even taking hours-long pauses.

source: University of Copenhagen

In a new breakthrough to understand more about the mammalian brain, researchers from the University of Copenhagen have made an amazing discovery. Namely, a vital enzyme that enables brain signals to turn on and off randomly, even taking hours of “breaks from work.”

These findings could have a major impact on our understanding of the brain and the development of pharmaceuticals.

Today, the discovery is on the cover of Nature.

Millions of neurons are constantly sending messages to each other to form thoughts and memories and allow us to move our bodies at will. When two neurons meet to exchange a message, neurotransmitters are transported from one neuron to another using a unique enzyme.

This process is critical for neural communication and survival in all complex organisms. Until now, researchers around the world thought that these enzymes were active at all times to continuously transmit essential signals. But this is far from the case.

Using an innovative method, researchers from the Department of Chemistry at the University of Copenhagen have scrutinized the enzyme and found that its activity switches on and off at random intervals, which contradicts our previous understanding.

“This is the first time anyone has examined these mammalian brain enzymes one molecule at a time, and we are delighted with the result. Contrary to popular belief and unlike many other proteins, these enzymes can stop working within minutes to hours. However, the brains of humans and other mammals have miraculous ways of functioning,” says Professor Dimitrios Stamou, who led the research from the Center for Geometrically Engineered Cell Systems at the University of Copenhagen’s Department of Chemistry.

So far, such studies have been conducted with very stable enzymes from bacteria. Using the new method, the researchers examined mammalian enzymes isolated from rat brains for the first time.

Today, the study is published in Nature.

Switching enzymes can have far-reaching consequences for neuronal communication

Neurons communicate through neurotransmitters. To transfer messages between two neurons, neurotransmitters are first pumped into tiny membrane vesicles (called synaptic vesicles). The bladders act as containers that store the neurotransmitters and release them between the two neurons only when it is time to transmit a message.

The central enzyme of this study, known as V-ATPase, is responsible for supplying energy for the neurotransmitter pumps in these containers. Without it, neurotransmitters would not be pumped into the containers, and the containers would not be able to transmit messages between neurons.

But research shows that there is only one enzyme in each container; when this enzyme shuts down, there will be no more energy to charge neurotransmitters into the containers. This is a completely new and unexpected discovery.

“It is almost incomprehensible that the extremely critical process of loading neurotransmitters into containers is delegated to only one molecule per container. Especially when we find that 40% of the time these molecules are turned off,” says Professor Dimitrios Stamou.

These findings raise many intriguing questions:

“Does turning off the power source of the containers mean that many of them are really empty of neurotransmitters? Would a large proportion of empty containers significantly affect communication between neurons? If so, would this be a “problem” that neurons have evolved to work around, or could it be an entirely new way of encoding important information in the brain? Only time will tell,” he says.

A Revolutionary Method for V-ATPase Drug Screening

The V-ATPase enzyme is an important drug target because it plays a critical role in cancer, cancer metastasis, and several other life-threatening diseases. Thus, V-ATPase is a lucrative target for anticancer drug development.

Existing drug screening assays for V-ATPase are based on simultaneous signal averaging from billions of enzymes. Knowing the average drug effect is sufficient as long as the enzyme works consistently over time or when the enzymes work together in large numbers.

“However, we now know that neither is necessarily true for V-ATPase. As a result, it suddenly became critical to have methods that measure the behavior of individual V-ATPases in order to understand and optimize the desired drug effect,” says the paper’s first author Dr. Eleftherios Kosmidis, Department of Chemistry, University of Copenhagen. who directs the experiments in the laboratory.

The method developed here is the first that can measure the effects of drugs on the proton pumping of single V-ATPase molecules. It can detect currents more than a million times smaller than the gold standard clamp method.

Facts about the enzyme V-ATPase:

See also

• V-ATPases are enzymes that break down ATP molecules to pump protons across cell membranes.
• They are found in all cells and are essential for controlling the pH/acidity inside and/or outside the cells.
• In neuronal cells, the proton gradient established by V-ATPases provides energy to load neurochemical messengers called neurotransmitters into synaptic vesicles for subsequent release at synaptic junctions.

About this neuroscience news

Author: Press office
source: University of Copenhagen
Contact: Press Office – University of Copenhagen
Image: Image is in the public domain

Original Research: Closed access.
“Regulation of mammalian brain V-ATPase by ultraslow mode switching” by Dimitrios Stamou et al. Nature

Summary

Regulation of mammalian brain V-ATPase by ultraslow 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 numerous cellular processes.

In neurons, the loading of all neurotransmitters into synaptic vesicles is activated 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 lifetime modes: proton pumping, inactive, and proton leak. In particular, direct monitoring of pumping revealed that physiologically relevant concentrations of ATP do not regulate intrinsic pumping rate.

ATP regulates V-ATPase activity through the proton pumping mode switching probability. Conversely, 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, which are expected to introduce stochasticity into proton-driven secondary neurotransmitter loading and may therefore have important implications for neurotransmission.

This work reveals and highlights the mechanistic and biological significance of ultraslow mode switching.