A marine biology project leads to proteins as ion channels

What work did you do for your Masters degree at the University of Alabama?

I wanted to get into marine biology, so I worked in the Biology Department with Professor David Kraus and his wife Jeannette Doeller on two very closely related marine crab species, Callinectes sapidus and Callinectes similis. (They are like your blue swimmer crabs here – which you actually get to eat, as well.)

These crabs live in an estuarine environment, where the fresh water from rivers comes into the ocean and meets the salt water, so they experience regular changes in the salinity of the water. Every time it rains and more fresh water comes in, the salt concentration changes. The two species have the same preying behaviour, what they eat. So why can one of the species survive in fluctuating salt concentrations, while the other one is much more restricted, needing a certain salt concentration and unable to survive in fluctuating salinities? That was my topic.

I found differences in the gill tissues with which the crab species breathe. Towards the end of my project it turned out that these tissues, which also regulate the salt concentration in the crabs’ body fluids, differed in the proteins that can form ion channels – basically a protein that goes across a membrane and can shuttle ions, charged particles, across. That lab was not set up to study ion channels, but after the end of this project I wanted to go on and learn, somewhere, more about those specialist proteins that form ion channels.

The PhD project: moving to ion channels in viruses

After you completed your Masters, you returned to Austria for a year to earn some money. Then you applied to do your PhD in a number of places around the world. You turned down an opportunity to work in Alaska, in favour of taking up a PhD scholarship at the Australian National University.

I had the offer of an Overseas Postgraduate Research Scholarship, but before deciding on it I came and visited the John Curtin School of Medical Research at the ANU to meet with my prospective supervisors, Professor Peter Gage and Professor Graeme Cox, basically just for a chat. I had originally applied for the scholarship because I knew they were doing ion channel research. But before coming around the world to do my PhD here, I wanted to know what they were working on and if I wanted to do that type of research. I was very fascinated by Professor Graeme Cox. His enthusiasm as a researcher really captured me and he was probably the reason why I decided to take up the project.

Professor Peter Gage is a neuroscientist whose group works on studying ion channels in the human brain. They’re made out of several sub-units and are very complicated to study. He is trying to work out exactly how the ions move across a membrane barrier through those channels. At the time that I joined, he had just started to look at proteins from viruses that also can form ion channels. They are much smaller proteins and the idea was, ‘If we can understand how the ion channels work in viruses, in a much simpler system, maybe that will give us a clue to how they work in the brain.’ So he had a student studying an ion channel from the influenza virus.

Graeme Cox is basically the biochemist of the team. He was going through several other viral proteins from all kinds of different viruses, deciding from similarities in their structure whether any could possibly also form ion channels. Out of the proteins on the list, I picked the virus protein Vpr, from HIV virus – I thought, ‘Well, if I work on viruses, HIV sounds really exciting.’ And having decided on that first trip out here what I wanted to work with, I then came back a couple of months later to take up my scholarship and start working on that project.

Linking ion channels to AIDS dementia: an exciting hypothesis

Why was your PhD work important?

It was very important because we showed that that particular protein of the HIV virus, Vpr, does form an ion channel – the first time any HIV protein has been shown to function like that. We went on to identify which part of the protein forms the ion channel, and then, because this ion channel’s characteristics were quite different from those of the ion channel in the influenza virus, we speculated that this one might be involved in the AIDS dementia of HIV patients. Many HIV patients in their late stages become demented and have problems with motor control and cognitive problems but it is so far not understood how this disease comes about and why it affects some patients and not others.

Since I have shown that this viral protein from HIV forms an ion channel, we have used neurons, brain cells, from rats to show that if this protein is present on the outside of neurons it can actually form an ion channel in the membrane of the neurons, and completely abrogates the normal functioning of the brain cells by interrupting the ion gradients across there. That is very exciting: if we can prove that that is taking place in the patients, perhaps drugs can be developed to alleviate the horrible AIDS dementia problems that they face.

Learning to study fully infectious HIV safely

What did you do after your PhD?

I returned to the US, to a different department of the University of Alabama at Birmingham. I worked in the lab of Eric Hunter, the director of the Center for AIDS Research. I basically went there because by the end of my PhD thesis work I was quite fascinated by how the HIV virus works but I had only worked with one part of that virus, one protein, and I had never come into contact with the entire virus. And to learn more about the entire virus I needed to learn how to work with a fully infectious virus. So part of my postdoc project in the US was to learn all the techniques that I needed for that. I learned how to work with all the proteins together in the live virus, under special physical containment facilities at what is called the PC3 level.

While I was there I studied a different protein of the HIV virus, a glycoprotein called gp41. It is on the outside of the virus, and it is very important in making the virus infectious. If the protein isn’t there or is truncated, the virus is non-infectious. And so it is interesting to study. Also, that particular protein has a very unusual long part on one end of it, and we made mutations in the protein by changing certain of its building blocks, to see what effect those changes would have.

People are quite concerned about the chance of contracting AIDS. Did you think about the risks of working with the fully infectious virus?

HIV is quite a safe virus to work with in a laboratory setting. The virus has a lipid bilayer around it. Normal bleach and detergents can actually remove that lipid bilayer and the virus is then non-infectious. Also, the whole training that goes with certification to work with the infectious virus is very strict. You work under very safe conditions. I feel that it’s much safer to work with it in a laboratory setting, where you know the risks and what you are working with, than it is in a hospital setting, where you work with patients’ blood samples that may contain all kinds of much more infectious viruses like hepatitis B and hepatitis C. So I believe that knowing the risk and actually controlling for it, and working under very safe conditions in those facilities, is quite safe – otherwise I really wouldn’t have done it!

Focus questions

• Piller investigates proteins that act as ion channels. What other functions do proteins have in living organisms?

• How does an accurate knowledge of the risks associated with a virus, such as HIV, help scientists study it safely?