6 May 2019

One of the gateways in the kidney cell virtually exclusively allows calcium through. But how does that calcium channel work exactly? What does the molecular machine look like that makes sure that the channel opens and closes? Radboud university medical center researchers and American colleagues have succeeded in charting this, using a new technique.

A human consists of roughly 100,000 billion cells. All those cells are relatively independent units, which generate energy within their own boundaries (membranes), construct proteins and perform countless other tasks. In order to do so, substances must be able to enter and leave the cell. At the same time, the cells keep in touch with their neighboring cells and the outside world. To achieve all that, cells have 'gateways' in their membranes. "Such gateways are crucial", says Mark van Goor. "In fact, without those cell gateways, life as we know it would be impossible." 

Hot peppers

A wonderful example of such gateways is that of the ion channels. These gateways allow ions - electrically charged substances - in or out. Van Goor: “They exist in the most diverse forms. For example, we know of ion channels which signal the burning of hot peppers in food or the hint of coldness in the menthol of your chewing gum. There are ion channels which register heat, or light, or ignition processes and so on. We still haven't charted the complete spectrum of the activities of all ion channels."

Calcium channel

Van Goor and Jenny van der Wijst, both affiliated with the Physiology department, have been working intensively for the last few years on one specific ion channel, the TRPV5 calcium channel. Calcium is a mineral necessary for the creation and maintenance of bones and teeth. It's effective in combating osteoporosis in older people and is needed for the correct functioning of nerves and muscles, blood clotting and the transport of other minerals in the body. Van der Wijst: “Most ion channels allow multiple ions through, but this TRPV5 channel specifically transports calcium. It plays a gatekeeper's role in the retrieval of calcium from the urine. In that way, the kidneys prevent too much calcium leaking away during the filtration of the blood and the calcium balance in our bodies is neatly maintained."

Nijmegen gateway

The Physiology department has a special connection to TRPV5, since it was discovered in 1999 at Radboud university medical center by Joost Hoenderop. Since then, much more has been learned about how this 'Nijmegen gateway' works. Van Goor: "For example, we now know that the hormone Vitamin D plays an important part in the regulation of the calcium. We know that the TRPV5 is actually always open, until the protein calmodulin closes it. And that all the other family members of the ion channel, such as TRPV1, TRPV2, TRPV3 and TRPV4 work in exactly the opposite way: they are always closed until opened by a special signal. 

Collaboration with US

"With our research from the last few years, we wanted to also chart the structure of the gateway, the molecular machinery behind that opening and closing", says Van der Wijst. "That only became possible through the revolutionary development of a technique for which the Nobel Prize was awarded in 2017: the cryo-electron microscope, or cryo-EM for short. We went to work alongside physicist Yifan Cheng of the University of California, who had the device - which costs around 5 million dollars - in his lab." 

Image in three dimensions

Van Goor and Van der Wijst, who both spent a considerable amount of time in the US in order to carry out the work on site, explain how the device works. “TRPV5 is an ion channel, a trans-membrane protein which acts as a gateway in the cell membrane. These proteins are added in large quantities to water, where they can move freely. We then freeze the whole and take several 2D photos of the proteins from all sorts of angles. We can then construct a 3D image from the thousands of 2D pictures. That's an almost film-like experience; amazing to get a picture of this ion channel as molecular machinery for the first time after at least a year of preparation!" 

On lockdown

Not only the structure of the ion channel was charted, the way in which the calmodulin protein 'locked down' the channel was also pictured. Van der Wijst: "If the concentration of calcium threatens to become too high, calmodulin binds to the ion channel and closes the gateway like a cork in a wine bottle. After that, the calcium concentration in the cell falls, the calmodulin is disconnected, and the calcium can enter freely again." 

Structure and function

Van Goor: “In the end, you want to chart the channel and the interaction with other molecules and proteins so accurately, that you can start using the computer to identify substances that could possibly function as medicine. That you can calculate the function based on the structure. It's called intelligent drug design. That would be a wonderful next step."

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