Image: ARNOLD ADLER The Rockefeller University
Roderick MacKinnon is no ordinary researcher. This 44-year-old physician turned scientist gave up a tenured position at Harvard--something for which many of his colleagues would no doubt give their eyeteeth--to pursue a problem he "just had to solve." It was no ordinary problem, either. In 1998 MacKinnon stunned the scientific community by revealing the three-dimensional architecture of a protein with paramount importance for transmitting electrical signals down nerve and muscle cells: a potassium ion channel. So significant was this discovery that it took the jury of the Lasker Foundation only one year to grant MacKinnon the Lasker Basic Medical Research Award. And this is no ordinary award: in the past two decades the majority of Lasker winners have gone on to become Nobel Prize winners.
On April 25, 2000, I met with MacKinnon in New York City at his Rockefeller University office overlooking the East River to ask about his work and his aspirations. The interview that follows falls into five sections. In the first part MacKinnon gives a mini lecture on the nature of electrical signals in living organisms, ion channels and the fascinating intricacies of their structure. He also explains the riddle of why potassium channels act like money machines that take nickels but not dimes. In the second part he touches on the hard labor he and his lab endured to identify the structure, as well as its potential applications for medicine. In the third part he voices his thoughts on the Lasker and the Nobel Prize and on his short- and long-term research plans. In the fourth part he discusses his past and divulges why he switched to a career in science after eight years in medicine. Finally, MacKinnon the trout-fisherman talks about how he likes New York and why he left Harvard and NIH funding for risky research projects.
SA: You have devoted your entire scientific career to studying ion channels. Why don't you start by telling me a little bit about why they are so interesting?
RM: There are many facets in the answer to that question, but one of the first things that fascinated people in general, even before they knew ion channels existed, were questions like "What is the basis for electrical signals in living systems? How is life electric? What is the electrical nature of living cells?"
A really obvious observation that people made early on is the very simple aspect of moving. I hold my hand out and I wiggle my finger. Obviously, a thought originated in my head--that I want to wiggle my finger--and then I wiggle my finger, and somehow there must be information getting all the way down to the muscles in my finger to make them wiggle, and that happens very fast.
Image: ROCKFELLER UNIVERSITY
It was understood early on, more than 50 years ago, that there are electrical signals that propagate down extensions of nerve cells called axons. And so a half-century ago, even before then, there was an effort to understand what is the nature of these electrical signals, how do they happen. [Alan] Hodgkin and [Andrew] Huxley, two scientists from England, came up with a theory for how it happens. Their theory was that the nerve extension, the axon, is like a cable, an electrical cable, where the cell membrane is the insulator around the cable, and the salt solution inside the axon is the conductor, as is the salt solution on the outside of the axon. And when they put their own and other people's information together, their theory predicted, among other things, that the cell membrane had to undergo changes in its permeability.
That is, the cell membrane first had to be such that ions, which are the charged atoms, don't cross the membrane very well, and then suddenly the membrane, in a little region, becomes very permeable. So first sodium goes across the membrane very well, and then shortly thereafter the membrane loses its permeability for sodium and then becomes permeable to potassium. This was a very central part of their theory. Since then people have tried to figure out what is it about the membrane that allows it to undergo its changes in ion flow, in its permeability. And people figured out that there had to be pores in the membrane, and those were called "ion channels."
I guess to answer the question, the very initial excitement about it was to understand electricity in living cells. And then the questions became more focused as people understood that there are channels through the membrane. They, and myself, wanted to know "How do they work?"
Different kinds of channels only let a specific ion through. So a sodium channel is called a sodium channel because when it is open, only sodium goes through and potassium doesn't go through. Sodium is a little spherical atom with a +1 charge on it, and the sphere of sodium is a little bit smaller than the sphere of potassium. They both have the same +1 charge on them. The sodium is 0.95 angstrom in its radius, and the potassium is 1.3 angstroms in its radius. So they are just a little bit different, and yet the sodium channel only lets sodium through, and the potassium channel only lets potassium through. So the question that I really wanted to understand is, what is the chemistry for this, what we call "selectivity?" How does the channel tell the difference between a potassium and a sodium? That's another exciting, fascinating feature for me that's driven my research.
SA: As far as I know, you found out what makes the potassium channel selective for potassium ions, as opposed to sodium ions. But why doesn't it let sodium ions through if they are smaller?
RM: When you consider what factors go into which ions go through the channel, there are really two sides of an equation you have to consider. The ions, like potassium and sodium, are very happy in water. So, for example, when you add sodium chloride (table salt) crystals to water, they dissolve very well in it. What's happening there is that the +1 sodium ion is separating from the -1 chloride ion, and they float off independently, or fairly so, in the water. What that means is ions, like sodium and chloride, are happy dissolved in water. And the reason they are happy dissolved in water is, in the case of sodium, that the water structure is such that it can organize around the sodium ion and stabilize it.
The sodium has a +1. And the water doesn't have a charge, but actually has a "partial charge separation." The H2O is like a little Y, where the oxygen is at the meeting point or the stem of the Y, and that's a little bit negatively charged. An equal amount of positive charge would be balanced over the hydrogens, the forks of the Y. So although it doesn't have a net charge, it has what we call a "little charge separation." The waters actually gather around, pointing the partially negative oxygen against the positive sodium ion, and we say the sodium is "hydrated" by water. If we could imagine seeing a sodium ion in water, what you would see is, the sodium would be in there, but then waters would be gathered around it, oriented with their oxygens toward the surface of sodium, so the partial negatives of the waters would be close to the positive charge of the sodium, since opposite charges attract, and that would be a stable situation.
Now, when you dissolve potassium chloride crystals in water, the same thing happens. The potassium and chloride would dissolve. Chloride is happy in water, and the positive potassium, much like sodium, is dissolved in water, with the oxygens pointed to the potassium. What this means is that these positive ions are happy in water.
To go into a channel, they have to come out of the water, and come into a little part that we call the selectivity filter. The potassium channel is such that it has oxygens, the same kind of atom as in the water, but they are part of the protein itself. And these oxygens are held, so that when the potassium comes in, instead of being close to the oxygens of water, it's close to the oxygens of the protein. Now, sodium can do that, too. But what the potassium channel does, it makes a hole that's a little bit bigger. It's a good size for potassium, and it's a little too big for sodium.
When we consider selectivity, let's think about sodium. Sodium can be in water, or it could be in the potassium channel. And where it's going to be depends on where it's energetically most stable. Now, in water, the oxygens from the water get very close to the sodium ion, but in the potassium channel, because the little hole that the channel provides is a little too large for sodium, the sodium would rather be in the water. And so what happens is it partitions or it stays in the water. The potassium, on the other hand, seems to be equally happy in the potassium channel and in the water, because the hole made by the channel is just the right size for potassium. And you have to understand, it's an energetic balance between "does the ion want to be out in the water?" or "does it want to be in the channel?" And to the best we can understand, this structure, of course, allows us to make this as a hypothesis. What it appears to us is that the sodium ion would rather be in the water than in the channel due to the hole size, whereas the potassium ion is equally happy in the channel and in the water, and thus you have a potassium channel.
SA: When you determined the structure of the potassium channel two years ago, was there anything completely unpredicted by previous mutagenesis studies or other studies?
Yes, there were many features that were really not predicted. People certainly had an idea that there would be a protein with a hole down in the middle, and in fact the idea that there would be probably oxygen atoms available to surround a potassium ion in the selectivity filter. And we even knew which amino acids would make the selectivity filter, we knew that from our own mutagenesis work in this lab. So we knew which amino acids would do it, but we did not know what kind of structure they would take. In order to understand that, we really had to see it, because there are so many ways you could arrange a given set of amino acids.
Then there was a feature of the channel that was altogether unpredicted. We never would have been able to predict it without seeing it. And that was this very strange arrangement of the helical segments of the channel. Proteins are made of some basic structural elements, alpha-helices and beta-sheets, and these elements were described a long, long time ago. It turns out the potassium channel is mostly alpha-helices, and it has a certain set of alpha-helices that are arranged in a very interesting way. The alpha-helix has polarity in that it has two ends; it is not the same going forward and backward. One of the differences in the ends is the amount of charge. One end that we call the amino terminus, the N-terminus of the helix, tends to be plus-charged--here again we are talking about charges--and the C-terminal end of the helix tends to be negatively charged. The channel's architecture is such that it points four helices, the negative ends of the helices, straight at the middle of the membrane.
And also, the channel's architecture is such that there is a cavity of water at the center. So when you look at the channel, what you realize is right at the point where the ion would be halfway across the membrane, there is a cavity of water, and the helices pointed with their C-terminal, or negative ends, towards the center of the cavity. In looking at that, you realize what the design is doing.
Again, this comes down to an issue of energetics. If you just think about bringing an ion from the water through a membrane, the channel somehow has to provide a pathway where the ion is energetically stable. One way it does it is by putting those oxygens that I have talked about in the selectivity filter. That's one part very near the outside edge of the channel. Right in the middle of the membrane, it doesn't have a selectivity filter, and that's in fact a place where, if you didn't have a special design, the ion, which is as we talked about most stable when it's surrounded by waters, is far away from the waters. So that's energetically unstable. But what the channel does is, it actually has a cavity of water in the middle, so it brought in water that stabilizes the ion at that point where it ordinarily would be farthest away from the water. So halfway across the membrane, there is a cavity of water, and then there are helices with their negative charges pointed at the cavity, and since positive attracts negative, that makes sense, because what it has is, in a sense, elements pointing partial negative charges to stabilize the cation [positive ion] at the center.
The cell membrane ordinarily would be a big energetic barrier for ions crossing the membrane, but the channel's design is such that this barrier was brought down, and the ion can easily slip through it. These features of the cavity and the helices were something that we never really could have predicted without seeing it.
SA: Generally, purifying proteins and then crystallizing them is an extremely tedious process. How long did this take you for the channel, and what was the most frustrating part of the process?
RM: Crystallizing proteins can be long and tedious, especially for what are called "membrane proteins," and ion channels are membrane proteins. Membrane proteins refer to proteins that are suspended in the cell membrane. Many proteins are floating in the water inside the cell, and those ones are generally easier to work with. The problem with membrane proteins is, their ends are pointed into the water on either side of the membrane, but the whole center of the protein is in the oily substance of the cell membrane.
In order to crystallize a protein, you have to take it and you have to first purify it in large quantity, and then you have to concentrate it and put it under conditions where it will organize into a crystal. Now, with a membrane protein suspended in the membrane, that's impossible unless you take it out of the membrane. I shouldn't say impossible; there is a technique where people make two-dimensional crystals, and they study those with the electron microscope. But the way we were approaching this problem is to make three-dimensional crystals, because if we could do that, we could solve the structure in a very straightforward way, if we could obtain good crystals.
The frustrating part is trying to produce good crystals. We figured out how to make a lot of the protein--we produced a lot in bacteria--but then the frustrating part is screening an enormously large set of conditions to find out which conditions would give us crystals of sufficient quality to solve the structure. Membrane proteins can take a very long time--many, many years--and we managed to solve this problem in about two years. But it was two years where we worked very hard indeed; many members of the lab worked hard on this problem, talented scientists like Declan Doyle, Joo Morais Cabral, worked for maybe two years; Alice Lee, Anling Kuo, Richard Pfuetzner, all of the authors on the paper worked hard over that two years. So in terms of person-years, it was many, many more than two years. We were all very excited about the problem, and we guessed that if we could see this structure, we could understand a lot more than we ever could without seeing it. So that's what drove us through this enormous set of conditions, and then many trips to the synchrotron--a great source of X-radiation that we used to study the crystals in order to deduce the structure.
SA: So studying the structure once you have got the crystals, is that a fairly straightforward process?
RM: Yes, fairly straightforward, although there still are challenges. The hardest part in this whole process is obtaining the crystal that's of sufficient quality. Once you have obtained that, it's fairly straightforward, but it can still take quite a bit of time. We had the crystals that were good enough to solve the structure almost nine months before we completed the structure, and it took us about that time.
SA: I think the structure you came up with revolutionized the whole field. Do you think it has applications to medicine?
RM: Yes, I do. People ask "Well, now can you immediately cure a disease with this?" and the answer to that is, no. It has applications to medicine in the way that it's laying the foundations for understanding an important class of molecules. So I can't say you can take the structure and immediately predict a drug that could then work on our own potassium channels, for example. That would be wrong to say.
But we need to consider what potassium channels do--they make the electrical signals in our hearts, in our brains; they aid our movements and our thoughts; they control the smooth muscle on the lining of our arteries, so they control blood pressure, and they also control the smooth muscles in our airways, so they can affect asthma. Because they play such a central role in many physiological processes in us, to understand the basic molecules that do this is merely a beginning to laying the foundation for eventual development of drugs that can affect some conditions.
There are certain diseases related to ion channels, but I would predict that the utility of drugs, of pharmaceutical agents acting on ion channels, won't be so much to take an abnormal ion channel and make it normal. I think it's more going to be to slightly affect the behavior of an already known ion channel, to basically correct a condition that's abnormal but not primarily related to the ion channel. So, for example, compounds that can affect the electrical activity of our nervous system eventually can be used as anti-seizure agents or anti-arrhythmics for the abnormal electrical activity in our hearts. Also there is potential for anti-hypertensives--blood pressure medications.
SA: Do you know of any pharmaceutical companies already trying to devise drugs that act on ion channels?
RM: There are many, actually. Many have contacted me for advice, for example. I usually only stay here in my lab and just concentrate on my own work, but that's my way. But yes, I do know that many are working on ion channels, in particular potassium channels as targets.
My understanding is that it will eventually work this way: You screen for molecules that will bind to a particular ion channel that you know is important to control some physiological process that goes abnormally--for example, a potassium channel in the nervous system that might affect the electrical activity of certain neurons. If you find a small molecule that can bind to that, through some screening assays and not direct use of the structure, you can then ask "Does that molecule affect an abnormal condition?" to help it. Once you know that, the molecule might have the desired effect, but it might not be good for treating somebody because of undesired effects or maybe because the drug can't be swallowed, so you would like to come up with a version that people could take as pills.
What you might want to do is ask "How is that drug working?" and ultimately then, you could solve a crystal structure with the drug bound to it. And then you can understand which part of the molecule is important for binding and having this effect on the channel, and which part is not. And then chemists can look at this and decide, "OK, well, this part is important for binding, so we need to preserve that, but this part is not. So if we want to change the properties of the molecule, so maybe someday a pill could be made out of this, this is the part we can work on, and this is the part we cannot." So that's the way this structural information I think will end up feeding in.
SA: You were awarded the Lasker Award in 1999, just one year after you had solved the structure of the channel. Your colleague here at Rockefeller, Gnther Blobel, received the Lasker Award in 1993, for work he did much earlier, in the 70s and 80s. Why do you think the jury members saw the significance of your work so easily? Why was it so obvious?
RM: I don't know [laughs], but I am very pleased that they did, to be perfectly honest. I feel very lucky that they did, and I can't really say why they did. I do know that in looking at the potassium channel structure, we could tell by some of the features that I've talked about (and, in fact, some that I haven't talked about) an enormous amount about the potassium channel.
One of the reasons the structure was so immediately interpretable was because ever since Hodgkin and Huxley published their work in 1952 on the theory of the nerve impulse, people have worked on ion channels and potassium channels, and they figured out a lot about the function of it. So there was a lot of imagination and a lot of nice thinking about what they ought to look like and how they might work. And then to finally see it was extremely satisfying. In some aspects, it was satisfying because many things were predicted, and in other ways it was satisfying because some things that were not predicted were almost more beautiful than anybody would have predicted. For example, the cavity and these helices were just a marvelous arrangement that Mother Nature used to solve this problem, you know, as if a very brilliant engineer did it all. I think that was very satisfying to see. Certainly it was to me, and I am happy that it seems other people appreciated it, but that's as much as I can say.
SA: You also know that Gnther Blobel received the Nobel Prize a few years later, and, in fact, 23 of the Lasker Award-winners won the Nobel Prize between 1980 and 1996. How do you feel about that?
RM: Well, you know (laughs), I would be very pleased, but I don't think about it. I am just concentrating on my science. I am already very pleased with how much people have seemed to have appreciated the work we have done here in the lab.
SA: What are you studying at the moment?
RM: Several things, and they are all an extension of the work we have done. One of the things we are trying to understand in more detail is: How do the ions go through the pore at the high rate that they do? The structure has allowed us to make initial hypotheses, but a higher resolution structure, combined with functional measurements, will allow us to test some of these hypotheses and understand the mechanism in more detail. That's one problem that we work on.
Another problem is, there are different kinds of potassium channels. The pore of all potassium channels that we know of is very similar, and what this structure has taught us is a great deal about how the ions go through. But channels also open and close--that process is called gating--and we still don't know very much about gating.
There is one particular and important kind of gating that is called voltage-dependent gating. Certain potassium channels open and close depending on what the membrane voltage is. That's an interesting thing because it is in fact the ion channels themselves that set the membrane voltage. So a potassium channel opens, and it sets the membrane voltage, and yet the membrane voltage also sets whether it's open. That kind of property is called recursiveness, so you would say the molecule is recursive: its action controls something, and that same something controls the channel, feeds back on it. We don't really understand how a channel can sense the voltage and open and close in response to it.
We know some things. From the work of many laboratories, we know the parts of the channel. So the voltage-dependent channels have an additional group of amino acids that is not present on the bacterial structure we solved. The pore, the channel part, the hole down the middle of a voltage-dependent potassium channel would be very similar to the channel we solved. It has an additional part that allows it to sense the voltage. We don't know what the structure of that is, and we don't know how that marvelous switch works. So that's one thing we focus a lot on in the lab, to solve the structure of a voltage-dependent potassium channel.
SA: How about the long-term future? Do you think you will always work on ion channels?
RM: I would say, I never know. I really enjoy working on them. I love working on them--but also sometimes, as a scientist, I find change good for me. I have been studying channels now for a long time. I began my postdoc probably in 1986 or 1987, that's when I entered science. I worked with Chris Miller at Brandeis for three years, and then in 1989, 11 years ago, I went on my own, and I've been studying channels for the first half of that using electrical kinds of measurements, studying the function of channels, and then for the second half of that, I adopted structural biology techniques, using crystallography to see what they look like. So that change, adopting new techniques, kept me fresh and happy and thinking about new things.
And then I suspect, if I had to guess, that I'll switch what I do eventually, that I'll think about something altogether new. It's not that I don't really enjoy studying channels, and there are problems right now that will keep me on channels. Some of these problems we just talked about that we're still working on, that keep me focused on them. But I think if we can solve these problems, I think I feel it would be a time for a change. I am not sure what that will be. I think it will always be biophysics, at the interface of biology and physics. That's what I like.
SA: If you could devise a magic machine that allows you to overcome the present limitations--technical limitations--what would you study? What questions would you like to solve if you had such a machine?
RM: The machine would be a way of producing crystals of the ion channels, very rapidly. That's a good question, and if I could have a magic one that would allow me to do that, allow us all to do that, it would be wonderful to immediately address some of these questions about, for example, how a voltage-dependent channel senses the membrane voltage. We could see a structure of one of those, and maybe its open and closed state. Then we would see, if [a part] moved across the membrane when it went from closed to open, that it carried some charges, and then we could immediately see how the membrane voltage can devise the conformational change of the protein. So I would like to have a machine that would allow us to very rapidly crystallize membrane proteins. That would be wonderful; a lot of people would love it.
In the long run, I don't know what machine I would want for the future, because I don't know what it is going be. But it's going to be something that's going to just grab me, one of these days, and I will want to know more about that.
SA: Let me ask you a little bit about your past, how you started your scientific career. How did you first become interested in science, and was there anybody who influenced you a great deal, say, before college and in and after college?
RM: I know I was always heavily leaning toward science and exploring things, even as a child. I was pretty curious about the way small things worked. I always liked puzzle solving; I liked to come up with an explanation to myself for how little things worked. But you know, it was nothing very directed. There were no scientists in my family. But I just know I loved exploring. When I was young I had a microscope, and I loved to peel blades of grass apart and actually look at the cells that the book told me should be there, and they really were. Or look at the microorganisms in pond water, and I found that fascinating when I was in elementary school.
I think when I really got turned on to science in a very serious way was when I was an undergraduate at Brandeis University. I really enjoyed my education there, and I think the person who has most influenced me is the person who subsequently became my postdoctoral advisor, Christopher Miller. But back in those days, when I went to college, he was my undergraduate advisor. He was a young professor, just setting up his lab, and you could tell he was really having fun at what he was doing, and I found him inspiring in his approach. I then went off to medical school, thinking I wanted to be a physician.
SA: I was wondering, why did you do that?
RM: Part of it was naivet. I thought medicine was a lot like science. I thought it was a branch of science. And it's really not; it's a special profession in itself.
SA: Do you think medicine will become more science-driven in approach in the future?
RM: I think it will be based more and more on science. It is based on scientific knowledge, and for that reason, as knowledge builds, the abilities of medicine, the effectiveness of medicine, will strengthen. But I think it will always be very different from science because, in a sense, in medicine you are usually working on a set of facts, and you have to know a lot. You have to be very good at pattern recognition and making connections, about what this set of symptoms could mean. But that's very different from puzzling over a really detailed problem, in being able to figure out how some very small thing works. It feels different; that's all I can say. Solving a problem in the laboratory that might have a theoretical component feels like you use a different part of your brain than when you practice medicine. At least that was my personal experience.
SA: Do you think that medicine has given you some kind of insight that a pure scientist might lack?
RM: I don't think so. I think it has given me a different view of things, but I wouldn't say that it has given me insights that somebody with a PhD training would lack, necessarily. I think it does give me a different perspective on it, but if I were asked, if I were to do it over again, I wouldn't go the medical route. Not because I regret it at all, I don't, but because I would rather have spent the time doing pure science for that period of my life. But I have no regrets at all.