Our cells move energy and matter to the places it is needed. But how do they do this in real time, and seen from the perspective of a single molecule? A Danish research team has successfully uncovered new basic insights into this invisible world, by doing experiments that track how a single molecule of the protein ‘engine’ known as the calcium pump works.

For the first time it has been possible to look directly at one of the most fundamental engines of the body: The ion pumps that power the cell’s transport and signalling systems. These functions underpin biomolecular mechanisms crucial to you, me and every other living creature.

The calcium-pump may not look like a lot. Each pump only measures a few nanometers, millionths of millimetres, in each direction, and sit in the cell membranes of our bodies. But despite its diminutive size, it is crucial to life. This pump is the reason that our muscles can contract, and that neurons can send signals. If the tiny pump stopped working, cells would stop communicating. Without it, we could neither move nor think. This is why cells use so much of their energy — about a fourth of the body’s fuel known as ATP — to keep the pumps running.

There are many things that we still do not know about the structure and function of this vital pump. Knowledge about the pump is essential to understand the energy balance and other important functions in the body.

A Danish research group has just published a new study that for the first time shows how the pump functions at the level of a single molecule, and how it ensures that ions are pumped in the right direction. In other words, how the pump works as a molecular one-way street. The discovery has just been published in the journal, Nature.

“This work represents the next step of a profound and important quest to understand the pump’s atomic structure and function. We are now one step closer to understanding how the ion pumps ensure the functions of the cells. We have characterized how it pumps ions out of the cell at an unprecedented level of detail. The importance of such basic knowledge of biophysical processes can only be underestimated. It will have a great influence on our understanding of the processes of life and, in time, on disease treatment,” says Professor Poul Nissen, head of both the Center of Excellence, PUMPkin, and the neuroscience center, DANDRITE. Prof. Nissen is one of the world’s leading experts on this family of pumps and a co-author of the paper.

The molecular backstop

To some extent, the story began in the 1950s, when Professor Jens Christian Skou did his pioneering work at Aarhus University, which uncovered the pumping functions in our cells. The calcium pump is a close cousin of the sodium-potassium pump Skou worked on, and they use a similar pumping mechanism. Skou’s work earned him a Nobel Prize in chemistry in 1997. Since then, numerous researchers have dedicated their working lives to uncovering the mechanism and function of these pumps — including many at the Center of Excellence for Membrane Pumps in Cells and disease, PUMPkin, at Aarhus University.

A key insight in the new publication concerns the one-way nature of the ion transport. Previously, it was assumed that the one-way nature of the pumping arose in the cleavage of the energy-rich molecule ATP. The hypothesis was that when ATP was cleaved, the pump could not backtrack and reform ATP. That turned out to be far from true:

“We have identified a new closed state in the pumping cycle, that the pump can only enter if the calcium ion comes from the intracellular fluids and the pump has cleaved ATP. It cannot reach this state if the ion comes from the cell’s surroundings. When calcium is released from this state, it is the ‘point of no return’. This is the mechanism that explains that the pump works as a pump and not just a passive channel.

This really unique insight is based on highly advanced experiments. These experiments enable us to directly see the pump doing its job for the first time,” explains Postdoc Mateusz Dyla, who is the first-author of the new paper, and who has been the main driving force of the project that started out as his PhD project.

The calcium-pump needs energy, which it gets from cleaving a molecule of ATP as explained earlier. The energy released is converted into the work of the pump. This explain how large concentration gradients build-up between the inside and the outside of the cell. The concentration difference can be more than 10,000-fold, and this large difference is essential to the communication between cells such as in nerve signalling.

Smoke and mirrors

The reason the experiments are so complex is pretty clear: The pump is so small that it cannot be imaged directly in a light microscope. So far, and with great difficulty, researchers have created molecular models of stable states of the pump using a technique known as X-ray crystallography. This is analogous to a stop motion movie. The scientists have jokingly referred to their visualisation of the pumps movement between these states as ‘Pump Fiction’. The new study, which has been five years in the making, moves the visualisation from stopped motion to live images of the functional movements of the pump. Technical improvement in microscopic techniques has allowed the new state to be observed.

The technique used is known as single-molecule fluorescence spectroscopy and use a phenomenon known as Förster Resonance Energy Transfer, in short FRET. Here, intense laser light and ultra-sensitive cameras are combined to allow the direct observation of a single molecule through the tiny amount of light each molecule emits.

The research group has taken advantage of a calcium-pump from the bacterium Listeria, which has been prepared for the studies through protein engineering. The engineering of the protein alone took several years to complete.

In the FRET experiments, two dye molecules are attached to the protein, which is then illuminated by laser light. One dye, the donor, will absorb the laser light and either emit it with a characteristic colour. Alternatively, it can transfer the energy to the other dye, the acceptor that will then emit light with another colour. Light will, thus, be emitted from the two dyes, and scientists can measure the distance between the two dyes by measuring how much light is emitted of each colour. Because the dyes have been carefully inserted in two specific positions in the pump, these distance changes track the pumping movements of the pump.

The single molecule technique is what has enabled the new discoveries explains Magnus Kjærgaard, fellow at Aarhus Institute of Advanced Studies, AIAS, who has also contributed to the discovery:

“We have moved from ‘Pump Fiction’ to ‘Pump Live’. Previously, we always recorded the signals from many molecules at the same time, which blurs the movements. Using single-molecule FRET techniques we can focus on one molecule at the time, which allow us to observe the structural changes directly. This provides us with a video of the pump in action with fewer gaps. Our Pump Fiction-movie initially got its name, because we knew the transitions between the different states of the cycle were fictional, and that there could be additional insights hiding in the gaps between the known states. We have now demonstrated this in abundance, and at the same time revealed critical new insights into how the pump works.”

Besides adding to our knowledge of the basic processes of life, the understanding of these pumps may also have practical applications. Mutations in the pumps can cause defects in brain cells, and this can cause neurological disorder such as migraine, temporary paralysis or neurodegenerative disorders.

The mechanisms of these ion pumps are thus pivotal to understanding the errors in the pump, especially with a view to developing new drugs targeting the pump.

“We haven’t reached the state where we can transfer our ion pumps research into treatment of disease yet! However, the new insights have led to ideas that may be used to develop treatment of for example defects in neuronal signalling. But this is work for the future. Right now, there is every reason to celebrate the revelation of the intimate details of one of the most important enzymes of life. The work has built on great collaborations here at the university, and with researchers in the US. We have already started new exciting collaborations that will allow us to take the next steps,” says Professor Poul Nissen.

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