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Major discovery about the mammalian brain surprises researchers

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Summary: V-ATPase, a vital enzyme that enables neurotransmission, can turn on and off randomly and even pause for hours.

Source: University of Copenhagen

In a new breakthrough to understand more about the mammalian brain, researchers at the University of Copenhagen have made an incredible discovery. A vital enzyme that enables brain signals turns on and off at random, even taking hours of “work breaks”.

These findings can have a major impact on our understanding of the brain and the development of drugs.

Today the discovery is on the cover of Nature.

Millions of neurons are constantly sending messages to shape thoughts and memories and let us move our bodies at will. When two neurons meet to exchange a message, a unique enzyme is used to transport neurotransmitters from one neuron to the other.

This process is crucial for neuronal communication and the survival of all complex organisms. Until now, researchers around the world thought that these enzymes were active at all times in order to continuously transmit important signals. But that is far from the case.

Using an innovative method, researchers from the Department of Chemistry at the University of Copenhagen have examined the enzyme closely and discovered that its activity is switched on and off at random intervals, contradicting our previous understanding.

“This is the first time anyone has studied these mammalian brain enzymes molecule by molecule, and we are amazed by the result. Contrary to popular belief, and unlike many other proteins, these enzymes can stop working for minutes to hours. Yet the brains of humans and other mammals are miraculously able to function,” says Professor Dimitrios Stamou, who led the study from the Center for Geometrically Engineered Cellular Systems at the University of Copenhagen’s Faculty of Chemistry.

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

The study is published today Nature.

Enzyme switching can have far-reaching implications for neuronal communication

Neurons communicate via neurotransmitters. In order to transmit messages between two neurons, neurotransmitters are first pumped into small membrane sacs (so-called synaptic vesicles). The bubbles act as reservoirs that store the neurotransmitters and only release them between the two neurons when it’s time to deliver a message.

The central enzyme of this study, the so-called V-ATPase, is responsible for supplying energy to the neurotransmitter pumps in these vessels. Without them, neurotransmitters would not be pumped into the reservoirs and the reservoirs would not be able to carry messages between neurons.

But the study shows that there is only one enzyme in each container; When this enzyme shuts down, there would be no energy left to drive the cargo of neurotransmitters into the receptacles. 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 realize that these molecules are turned off 40% of the time,” says Professor Dimitrios Stamou.

This shows a v-ATPase on a synaptic vesicle
The cover picture shows vacuolar-type adenosine triphosphatases (V-ATPases, large blue structures) on a synaptic vesicle from a neuron in the mammalian brain. Image: C. Kutzner, H. Grubmüller and R. Jahn/Max Planck Institute for Multidisciplinary Sciences. Photo credits: C. Kutzner, H. Grubmüller and R. Jahn/Max Planck Institute for Multidisciplinary Sciences.

These results raise many exciting questions:

“Does turning off the containers’ power source mean that many of them are actually devoid of neurotransmitters? Would a high proportion of empty bins significantly impair communication between neurons? If so, would that be a “problem” that neurons were designed to work around, or could it possibly be an entirely new way of encoding important information in the brain? Only time will tell,” he says.

A revolutionary method to screen drugs for the V-ATPase

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, the V-ATPase is a lucrative target for the development of anticancer drugs.

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

“However, we now know that neither of these things necessarily applies to the V-ATPase. Therefore, it has suddenly become crucial to have methods that measure the behavior of individual V-ATPases in order to understand and optimize the desired effect of a drug,” says the first author of the article, Dr. Elefterios Kosmidis, Department of Chemistry, University of Copenhagen, who led the experiments in the laboratory.

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

Facts about the V-ATPase enzyme:

See also

This shows a diagram of the gut and brain
  • 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 pH/acidity levels inside and/or outside 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 news from neuroscientific research

Author: press office
Source: University of Copenhagen
Contact: Press Office – University of Copenhagen
Picture: The 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


abstract

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 set up electrochemical proton gradients for a plethora of cellular processes.

In neurons, loading of all neurotransmitters into synaptic vesicles is stimulated by approximately one V-ATPase molecule per synaptic vesicle. To shed light on this true 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 over time, as suggested by observing the rotation of bacterial homologues and assuming strict ATP-proton coupling.

Instead, they stochastically switch between three ultra-long-lived modes: proton pumping, dormant, and proton leak. Remarkably, direct observation of pumping showed that physiologically relevant ATP concentrations do not regulate the intrinsic rate of pumping.

ATP regulates V-ATPase activity through the probability of switching the proton pump mode. In contrast, electrochemical proton gradients regulate the pumping rate and the switching between pumping and inactive modes.

A direct consequence of the mode switch are all-or-nothing stochastic fluctuations in the electrochemical gradient of synaptic vesicles, which might be expected to introduce stochasticity into the proton-gated secondary active loading of neurotransmitters and thus have important implications for neurotransmission.

This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode switching.

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