Function and structure of ion channels

Kandel: This was not the era in which Alex Mauro taught at Yale, was it?

Hille: Well, he was actually at the medical school at that time. And he was not teaching then. I think he was more or less taking care of electronics for Fulton and his crew. Setlow and Pollard were people who were at Yale at that time and had run the biophysics. And Harold Morowitz was an important person at that time. So through zoology, I got a love of, for example, evolution and learned the taxonomy of lots of kinds of organisms, something that always interested me and accounts for why at all times I seem to speculate about evolution of ion channels and what organisms used them for what.

So then I think we could go to Rockefeller. The reason I went to Rockefeller was that Ed Boell, who had taken me into his laboratory, wrote to Paul Weiss, who he knew, and Detlev Bronk, who was the president of Rockefeller, that apparently that I was an okay student and I should go there. So he told me about this letter, and I had never heard of Rockefeller, so I knew nothing about this. I was invited down to Rockefeller and Detlev Bronk spent the whole day, the president, with me while he was talking to David Rockefeller and running the National Academy of Sciences and running the university, all at the same time. And he spent the whole day with me, and then he said, okay you can come. And I said, well, I have to think about it. And when I came home Ed Boell said, what are you trying to do, play hard to get? (Laughter.) So basically I never applied anywhere else. I never applied anywhere. I went there because more or less my mentor said that is what I should do—which was terrific.

Kandel: Now this was the beginning of the graduate program at Rockefeller, if I remember correctly?

Hille: Well, it had been going for maybe five or six years. So there were a number of classes before me. In the earliest class were people like Gerry Edelman and Ed Reich and other people. So I was maybe the sixth class. This was actually a really unusual place, and it may not be quite the same anymore, but at the time Bronk had a very stylish way of having everything run. Everything was the best. The students had a very large budget, an infinite budget basically, and you could just go down and order on 8202, which was the budget number. Everybody will remember 8202. Whatever we wanted. Bronk called the Rockefeller a “community of scholars” and said that we were part of the community of scholars, so at once we were no longer students, we were just part of the community of scholars. And we always heard about many great people and the many Nobel prizes. And I took classes with George Palade and people like that. I did electron microscopy with him. And so we really were privileged.

Kandel: Who was in your class?

Hille: Well my class… there were 20 people in my class and six of us became members of the National Academy of Sciences. My closest friends were, for example, Harvey Lodish, who does cell biology at MIT; David Sabatini, who does cell biology at NYU; David Hirsh who is chairman of your biochemistry department.

Kandel: These are wonderful people.

Hille: These are examples of people who I, almost within the first week, became closely associated with, and our group was very highly intellectual and had a lot of different backgrounds. Harvey was a chemist, for example, at that time. He knew nothing about biology, so now he was supposed to study biology. So I gave him an enormous book of histology, which was by Ham and Leeson, I think. I gave it to him one night, and the next morning he came in and said, “That was a pretty good book. Do you have another?” (Laughter.) That was kind of the caliber of people who were there.

Kandel: Was Chuck in that year?

Hille: No. Chuck Stevens was maybe three years ahead of me. He was certainly there. Alan Finklestein was maybe three or four years ahead of me. Fred Dodge was perhaps five years ahead of me. But all those people were there still, and I knew them quite well and interacted with them.

Kandel: How did you focus in on your research project?

Hille: Well, interesting. I forgot to mention that at Yale, I was assigned a sort of senior honors advisor whose name was Tim Goldsmith, who a bit later became the chair of zoology at Yale. And so Tim (this was his first year at Yale too) didn’t talk to me much, but every once in a while would slip me a note and say, try reading this interesting paper. And then we didn’t talk about it afterwards, except he would say, “How was it?” And I would say, “okay,” and then he would give me another slip of paper. One of them was the Hodgkin and Huxley papers. So he just sent me off to the literature to read five papers by Hodgkin and Huxley. We never discussed it. It was kind of amusing. And I said it was okay.

And then he would give me a paper, let’s say in German, on the liquid junction potential by Henderson, and it would be from 1924. And I’d go off and find that somewhere and read that and say it was okay. Real eclectic, interesting stuff, which made me know the Hodgkin and Huxley papers before I came. In fact, Kacy Cole had come as a lecturer at Yale. So I heard him give several lectures which were really on the squid axon, including the Hodgkin and Huxley stuff. So by the time I came to Rockefeller, I already had some exposure to that. And I had, through my zoology training, sort of come to the conclusion that all of this was not really that interesting, that it wasn’t so interesting for biologists because it was a whole bunch of numerical and empirical curve fitting—events that biologists didn’t seem to be very interested in. And probably I should do something biological.

So I sort of rejected this area. But then I decided, well, membranes were really still interesting. So I tried to work on the sodium potassium ATPase, and I did that in Alfred Mirsky’s lab. And then in Palade’s lab, I studied holes made in red blood cells by complement, making lysis. And I did a variety of projects like that, but the people I was working with never got interested in them. So eventually I went back to being interested in the axons and their electrical excitability because Alex Mauro said, “Well, you know, if you don’t have anything else to do, why don’t you come to our lab, and you can study the effect of D2O on the propagation of impulses in lobster nerve trunks.” And that was great. We got a lot of lobsters and had a lot of bouillabaisse and a lot of parties, but it wasn’t really getting very mechanistic. Now this laboratory was the laboratory of Detlev Bronk, so it was the President’s laboratory. And it was peopled by Frank Brink, who was the dean of graduate studies; Clarence Connolly, who was the associate dean; and then Alex Mauro, Paul Hurlbut, and—I guess those were the people who were there.

So basically it was mostly an inactive laboratory. Of course it was all the deans and the president. The president never came, and the deans didn’t do any research, and Alex Mauro and Paul Hurlbut were doing some things. And Alex was, as you know, an eclectic person who had an interest in muscle satellite cells, in negative pressures, in osmosis, in thermodynamics and electrochemistry, in the history of Galvani and Volta and black widow spider venom toxins and later in Limulus ventral eye photo detection. So he had a lot of interests… it wasn’t any particular one thing that he focused on. And it was in that laboratory that Fred Dodge had done his voltage clamp work on nodes of Ranvier. And he had graduated just the year I came in and was now actually in Keffer Hartline’s laboratory studying the Limulus eye, which was on the same floor on the same hall. And Alan Finkelstein had just finished his work, and Chuck Stevens was graduating.

So Fred Dodge said, “You know, if you don’t like the D2O on the lobster axons, why don’t you use my old voltage clamp, which is sitting over here? We haven’t used it for several years, and it’s covered with dust, but we can make it go.” So he helped me fire up this machine with a whole rack full of vacuum tube power supplies and 300 volts positive and negative, and wound up all these things. There were a lot of things broken, and he just went in one after the other and fixed each part and set up things. And I gradually began to feel that if you were interested in electronics and you had to use it, you didn’t have to be some electrical engineer to fix it because here was Fred Dodge, who had just come from New Hampshire and wasn’t an engineer, and he was enjoying it, and it was very rational. So I got from this example the idea that one could work on electronics. In fact, I had taken my general exam at Rockefeller and one of the outcomes was that if I wanted to go into neurobiology, I better learn something about electronics.

So I learned a lot about that and from Fred’s lesson, I had actually learned a little bit of Fortran programming when I was at Yale and did some Fortran programs for the Hartline laboratory. In exchange they taught me how to do machine language programming, which we could do at the bit and byte level on their computer, which filled a whole room. They had a laboratory computer, one of the early ones, that took a whole room of space and had, gosh, I can’t remember, maybe 8,000 memory locations in it or something like that, and big magnetic tapes. So I learned from Fred Dodge how to voltage clamp nodes of Ranvier. And I photographed the pictures on graph cameras, as people did in those days, and projected the pictures under an enlarger on graph paper and tried to analyze the kinds of numbers that Hodgkin and Huxley were able to get and Fred Dodge and his mentor Bernard Frankenhaeuser in Sweden with whom he had done his original work. I got quickly bored, or discouraged, by how much projecting of film and measurement you had to make in the dark. And I decided that maybe you could automate this and use this computer which Hartline had in the other laboratory down the hall.

Part 3: The Saturday Project
Hille does all the experiments for his thesis at Rockefeller on Saturdays, the only day he was allowed to use the computer he needed for his work.

Kandel: So let’s stop here for a moment. Hartline was presumably not using it for online analysis. He was using it for data analysis after the experiment.

Hille: They actually were making online recordings. Their computer was new when I came, and you couldn’t buy an A to D converter and a D to A converter to make the recordings. They were designing them and building them. And they actually in the end put in sinusoidally modulated light into the Limulus eye and measured the voltage in the Limulus photoreceptor, which became sinusoidal. And these were recorded by the computer. And then they injected sinusoidal currents into the photoreceptor and measured the spike train that came out, which also had a sinusoidal component. They measured basically what was called the transfer function of each of the stages, and then they showed how as a linear system it operated. That’s the kind of thing they were doing. Actually what I wrote for them in Fortran was cross correlation analysis for them to cross correlate the sinusoidal inputs and outputs. But no one had ever done voltage clamp with a digital computer at that time.

Kandel: So this is the application based upon physical analyses.

Hille: The fact that the computer was there was perfect. It actually belonged to Hartline, and I was only allowed to use it on weekends. I did all the experiments in my thesis on Saturdays. And even when I came here to the University of Washington, I did experiments on Saturdays because the computer I was able to use here had the same deal. What I set up was a way to stimulate the node and record both the voltage and the currents and then to later subtract, or measure, from the records the leak current and subtract that, and measure the shape of the potassium current and fit that with the function, and then subtract that and leave the sodium current and fit it to rising and falling functions of the Hodgkin and Huxley type.

Kandel: I just want to step back for a second. If I remember the Dodge and Frankenhaeuser paper, they essentially confirmed for the node the Hodgkin and Huxley analysis of the squid axon, with the exception, I forget, there was no inactivation or there was less inactivation. I’ve forgotten the details, but it was pretty much showing that really a vertebrate nerve functioned in the same way as the squid axon. What specific question did you begin to address?

Hille: So right away… I just looked at my notebooks in preparation for thinking about this to see what I did. And the first experiment I did was to put on local anesthetics, Xylocaine (Lidocaine). And the next experiment was to put on tetrodotoxin, and the next experiment was to put on tetraethylammonium ions. And this was node number one and node number two and node number three.

Kandel: Isn’t that terrific! So maybe, for the person who’s not familiar with this, maybe you put this into some sort of a theoretical…

Hille: Why was I doing this? Well, I was in the laboratory of Alex Mauro, where he and Alan Finkelstein had been writing papers about electrodiffusion. I wasn’t clear exactly what they were saying, whether they were saying that they thought that all kinds of different ions could sort of go through the same regions of the membrane and they were showing how you would calculate the voltage and currents in that circumstance. And I thought they were maybe saying that in axons all the different ions were going through the same holes, not even holes, but just kind of the membrane itself. Like a piece of Kleenex, without any structure implied. And I personally thought that the currents carried by sodium ions and those carried by potassium ions must be going through different places.

Kandel: On the basis of what?

Hille: Well, so we sort of skipped over what we knew at the time. The Hodgkin and Huxley analysis had described the time course of an increase of sodium permeability and then its fall and then the time course of a rise of potassium permeability. And they declared that they couldn’t say anything about the structure of the membrane from this, but they could fit those time courses with mathematical functions and show that anything with those properties would be able to make an action potential because the rise and fall of sodium permeability and the rise of potassium permeability are exactly what you needed to make an action potential. So they had basically explained how you make the electrical signal of an axon—how you make it propagate—but had declared that no structural conclusion could be drawn from that. And now I think they felt, but they were reserved enough never to declare ever, I think they felt that most likely there are separate places for the sodium ions and potassium ions to go. A sodium-carrying mechanism and a potassium-carrying mechanism, which seemingly we began to call sodium channels and potassium channels. And that was sort of the issue with my perception of what Alex Mauro and Alan Finkelstein had done. It sounded to me like they were saying maybe these things go in the same place, and there certainly were many biophysicists who were saying that.

Kandel: Mullins… many people.

Part 4: Early Experiments
Hille details his work on axons that results in the first convincing demonstration that there are separate conduction pathways that can be molecularly distinguished from one another.

Hille: Mullins, in particular—a person who kept being the Devil’s advocate. He had nothing to lose, and he was a good protagonist for his point of view. He always pointed out the defects in the arguments. So it was sort of in that context that I felt that you could use pharmacological approaches or chemical approaches and decide that, if you could block or eliminate one of these two things without affecting the other, that they must be separate mechanisms. Hence my experiments with local anesthetics and tetrodotoxin and tetraethylammonium ions. Tetrodotoxin had already been described by Narahashi and Moore as blocking sodium conductance. The other ones were just sort of coming out of the blue. Clay Armstrong had a paper with Binstock in ’65, which was there first… on tetraethylammonium. My work was actually started before that. It came out after that. But tetraethylammonium ion and local anaesthetics and a long list of many other things had been in the literature as affecting conduction and shape of action potentials. And so what I did was to go back to—there was an enormous set of reviews, for example, by Abraham Shanes.

Kandel: I knew him well. Abe Shanes. Wonderful review.

Hille: Yeah, 140 pages long.

Kandel: Yeah, but terrific.

Hille: And he collected the effect of every drug that was known to work on axons. And so I just went through the list and I actually probably tried 100. And of those, you know, I happened to start with good ones right at the beginning. So those were my first experiments.

Kandel: But that must have been just a wonderful result. Did you and others appreciate how awesome the conclusions were that you could draw from that?

Hille: Well, immediately I declared it in my papers. The papers would always start out by saying—my papers—that we could block the sodium current with this and then the potassium current with that. And then when you got to the discussion, I would say, therefore, now we have separate places and we are going to call these sodium channels and potassium channels. That was the standard structure that I had. That to me was an important conclusion, and many people didn’t believe it, so it was still a big issue at that time.

Kandel: But that was probably the first convincing demonstration that there were separate conduction pathways that could be molecularly distinguished from one another.

Hille: Yeah, you could say that Narahashi and Moore in ’62, who had used the tetrodotoxin, you know they already had tetrodotoxin blocking sodium. And I showed it both ways. They actually didn’t believe it so much, so sometimes later they would publish a paper with an agent that blocked them both, and then they’d say, well, maybe they’re together. It wasn’t something that they firmly believed in. Clay Armstrong certainly believed in it already, and I believed in it already, before we started any experiments.

Kandel: Did you also conceive of the channels as being protein molecules? Was that clear at that time?

Hille: It wasn’t, no. An important set of classes that I had taken at Rockefeller was from Dan Koshland who was there. Actually he was at Brookhaven.

Kandel: Yes. He taught as an adjunct at the Rockefeller.

Hille: Yes, that’s right. So I took a course of theoretical chemistry from him, which I thought was terrific. And then I took enzymology from him, and that was terrific. So I had already had this training, which said that you could learn about the active site of an enzyme.

Kandel: And he was also very much interested in shape changes and allosteric mechanisms and conformational changes.

Hille: Exactly. Structure-activity and all those things. It was very clever. And how through chemical thinking, you could prove things. So I wanted to emulate that. This was a model for me even before I began experiments. These were things that I had heard about, and I said we’re going to do this to what’s in the membrane. But looking at my entire thesis, for example, I don’t think I ever used the word protein. And at the end of the thesis, there’s a drawing, which actually is going to be reproduced in Nature Medicine, drawing separate sodium and potassium and leak channels in the membrane and also an enzyme and also the sodium potassium ATPase—the only things we knew in the membrane. And they’re just sort of like little funnels, with nothing sticking out of the membrane, just the membrane kind of makes a funnel inside of it. There’s no substance. There’s no material, no chemical there.

Kandel: You know the discussion between Crick and Hodgkin that I think Hodgkin in his autobiography or Crick in his autobiography mentions, in which Hodgkin asked Crick, what kinds of molecules might mediate these currents? And he said, it has to be proteins! (Laughs.)

Hille: Now, I think if you asked me at that time, I would have said exactly the same thing. And certainly by the time I started my work in Seattle in 1968, I had proteins in my mind. But looking at my thesis I don’t see evidence that I really said proteins. I was telling you about Hodgkin, since I worked with him for a while and was associated with him. He said that you couldn’t write or talk about what you did when you were younger without actually going back and reading what you wrote down at the very same time, because you would always make yourself look better.

Kandel: Right. (Laughs.)

Hille: So I have to confess that I can’t say protein. It didn’t take many years though.

Kandel: Although the concept of channel had been around, you were the first one to actually use specifically sodium and potassium channels.

Hille: Actually in the literature and in my book, I mention about four papers in which the word appears once in somebody’s writing.

Part 5: Post Doc in England, On To Seattle
Hille works with Alan Hodgkin in England then finds a post at the University of Washington.

Kandel: As sodium and potassium channels? You trace the word channel all the way back to the 19th century, but I thought that—well, you tell me, go ahead.

Hille: In the Cambridge Group, Cambridge, England, which would be Hodgkin, Huxley, Keynes and those people. For example, in the Hodgkin and Keynes paper, which was 1955, about “it must be that the potassium channel has lots of ions in it in a row” they used the word channel once. They don’t say potassium channel, I don’t think. So it could be that Clay and I were… Clay certainly used potassium channel right away, without question. He started his terminology from the beginning of the paper. I would always be conservative at the beginning of the paper, and at the end of the paper I would be… “now we are going to call them… ” But he would just start right out. So he was clearly leading me there. Okay, so I think now we’re finished with my thesis basically, which was at Rockefeller, and maybe by now we’re… I had a year in England.

Kandel: That was right after Rockefeller?

Hille: Yes, I was postdoc with Hodgkin for one year.

Kandel: Now tell me what year that was.

Hille: That was 1967-68. And at that time Alan Hodgkin was doing experiments with Mordy Blaustein on the sodium-calcium exchanger. So I went to Plymouth, he sent me to Plymouth, and I was to study the calcium action potential that Tasaki had reported, that is an action potential carried by calcium ions. And I failed utterly to be able to do this. I was allowed as equipment only two cathode followers. He told me I couldn’t voltage clamp, and I wasn’t allowed to perfuse the axon. So I felt rather crippled.

Kandel: Why did he place those restrictions on you?

Hille: Well, he had the impression that experiments were very hard. And so, for example, when I was a graduate student after the internal perfusion of the squid axon was published by Baker, Hodgkin and Shaw. I wrote a letter to Hodgkin, after I had learned how to voltage clamp, saying this seemed like a wonderful technique, and the idea of combining perfusion of axons with voltage clamp would be wonderful. And actually I have a letter right here in front of me. He wrote back and said, “The technique is formidable, and I rather doubt whether anyone in Plymouth will get it going again in the immediate future.” Actually he had told me that Knox Chandler and Hans Meves were doing that technique at this very moment, as he was writing this letter, in Plymouth. But then he was telling me that it was too hard to ever do again. So somehow he had the impression that experiments of that kind were so difficult that one couldn’t do them. Interesting. Kenneth Cole told me when I wrote him that I was going to have a year with Hodgkin, he said, “I should warn you, Hodgkin doesn’t like voltage clamps,” and he was right.

Kandel: That’s amazing. It’s absolutely amazing.

Hille: And it could be some kind of caution, that he thought that the experiments required a genius like Huxley to be there to be sure that they were done right. And that if you did something, you would probably do it wrong, and therefore, it wasn’t worth doing. I’m guessing that that was sort of the context of this. Larry Cohen was actually in Plymouth at the time beginning optical experiments, and he had found a birefringence change, and I had actually made a voltage clamp surreptitiously for the squid axon, even though I was told not to. And Richard Keynes heard about that. So he said, you know, why don’t you stick your voltage clamp into Larry Cohen’s axons and we can do birefringence with voltage clamps. And I switched over to doing that. And I continued actually that year working mostly with Larry Cohen in Richard Keynes’s place. So I spent very little time with Alan Hodgkin. Actually when I finished at Plymouth, Alan Hodgkin told me that there was no place for me to work in Cambridge, so I would have to do a theoretical problem. That was one of the bases of my continuing in Babraham, because I had no theoretical problem in mind what so ever.

Kandel: So you came back to the United States in ’68?

Hille: Actually, while I was a graduate student, before I had defended my thesis even, I had made a tour of the United States, especially the West Coast, looking for jobs, and I went to maybe seven institutions on the West Coast. And I liked Seattle, University of Washington, the best. Several places gave me offers.

Kandel: You told me once I think, many years ago, that one of the reasons you liked Seattle is because Woodbury had computers. Is that right?

Hille: It’s exactly right. Because I had more or less decided that it was necessary for me to have an online computer to record my experiments or else I couldn’t do them. And it wasn’t actually Walter Woodbury who had them, it was another person who was making big computers in the department, but they were here and they knew all about what that was.

Kandel: And they committed themselves to you that you would have access to it.

Hille: Right. On weekends. (Laughter.) And then I could also use them for an hour or two here and there to analyze. But to do experiments you have to have a lot of time. So I did those on the weekends, too. So really, you’re right, it was because they had computers. When I did this trip—interesting what those days were—it wasn’t because any place had an advertised job. It was because Alex Mauro had written to these places and said, “My student is coming by, and he’d like to give a talk. And he’s looking for a job.” Several places wrote back and said they didn’t have jobs, but I could come. And I wrote Chuck Stevens, who was here, because I had known him as a student—and the same thing, he said no job. So I went to all these places and almost every one of them offered me a job. So in those days, jobs just materialized if they had somebody in mind, and otherwise there was nothing. You didn’t have to advertise or compete, it was just a matter of did they want some such person. Nobody paid my airfare or trip expenses except for Simon Fraser University in British Columbia, which paid the airfare from Vancouver, British Columbia, back to New York. Nobody promised me any startup money. So I accepted the deal in Seattle without their having paid any of the expenses—I actually stayed in Chuck Steven’s house—without any startup money; no statement about how much teaching or space I would get, but I would get a room somewhere. And you just shake hands on that, and that was it.

Kandel: You should negotiate all over again. (Laughter.)

Hille: Yeah, okay. So when I came here, the first thing I decided to do was to measure the conductance of single channels, or to work on that problem, which was a problem which wasn’t really solved until patch clamping came out, although noise did something good for it. And I spent a frustrating year working on that question, and finally just threw out everything.

Kandel: So I’m sorry, this is something I was not aware of…

Hille: Because it’s not published.

Kandel: That I’m aware of (laughs). But how did people think about single channels in ’68? Did they think about it?

Part 6: Related Studies
Hille comments on papers by other scientists related to his work on composite currents.

Hille: Well, in my thesis in ’67 and in a paper I published in ’68 as an appendix, I calculated the conductance a single channel could have on the basis of diffusion to the channel, on the basis of resistance that a hole would have if it was small enough to hold an ion, and things of that kind.

Kandel: So, were you the first one to begin thinking about the fact that these were composite currents and that one could think of them as being made up of lots of channels?

Hille: Well I don’t think so, but I actually believe I’m the first one to try to write down numbers in the literature that said, this is what the conductance of a channel might be, and this is, therefore, how many channels you might have in a node of Ranvier because of that.

Kandel: How did this relate to the period in which people were doing artificial membranes?

Hille: Okay, well yes, that’s a good point you’re making. Hladkey and Hayden actually were working on gramicidin. I met Stephen Hladkey in Cambridge when I was a postdoc, and they were just starting those experiments. So probably by the time I came here they had published a single channel gramicidin paper in bilayers—in ’68 or something of that time. Also Katz and Miledi had begun to…

Kandel: But they didn’t publish until the 1970 or 1971. The noise analysis. And they began to think of that for the ACh receptor, sure.

Hille: But my writing about the conductance was probably before that stuff. And I think I was a factor of three off. What I calculated mostly was the limiting possibilities. You can’t go higher than 300 picosemens, I said, and that’s about the highest channel that we know of even today. And it turned out that sodium channels are somewhat smaller. So anyway, I tried to do this, and I was completely fooled by… It turned out if you looked up on a random number table, you could get data that looked like the data I had. And if you binned them into histograms you would see periodicities that would make you think that you had sort of modes of their stepwise increasing of an electrical property or any property that you measured. And I was completely fooled by what you could find in a random number generator and making a histogram from it—that you get periods. I was looking for the effects of 1 channel, 2 channel, 3 channel, 4 channels kind of thing. And so when I finally had the sense to use a random number generator to compare my experiments with, I realized it was junk. So then right away, the next day, I looked in my notebook and it says, “The conclusions from this set of experiments are”—and then in red—”zilch.” The next day I mixed five different ions in solutions and began to study selectivity, and that worked.

Kandel: So this was clearly in the back of your mind.

Hille: And it was also in my grant proposal. In fact, I had even done some as a graduate student. As a graduate student perhaps the best paper that came out when I started was the paper by Chandler and Meves, where they had done this work that Hodgkin said was going on in Plymouth—perfusion—and they showed that the sodium channel of the squid axon can accept lithium, sodium, potassium, rubidium and cesium and gave permeabilities for each of those. So soon after that, I did the same experiments, a little bit, myself at Rockefeller. For example, lithium is published in at least one paper of mine. So I knew that you could do it, and I did this in earnest at the University of Washington.

Kandel: If I remember correctly the Meves-Chandler paper did not use organics. They used a very limited range.

Hille: They just used those five. Now in the literature there had already been papers by Koketsu and Tasaki.

Kandel: They tried everything—in some haphazard way.

Hille: And all of these authors had shown that if you take away the sodium…

Kandel: You could substitute.

Hille: You could give it guanidinium or something else.

Kandel: They did it in order to make Hodgkin look foolish.

Hille: That’s right. So their purpose was to refute the sodium theory, and my purpose was to take their interesting results and interpret it as those ions going through sodium channels, which I did. Actually somebody, I think Ehrenstein and Gilbert at the NIH, had already published a paper, I think at that time, showing that ammonium would substitute for potassium in the squid ion axon, perhaps in the potassium channel and maybe also in the sodium channel, and from those kinds of experiments then they made some speculation about how selectivity works, because ammonium has a four-fold symmetry or a tetrahedral symmetry or whatever. And I decided that you couldn’t really do it on the basis of just one other ion, you had to take every possible ion, every ion that existed and see what the results were before you could sit down and make what your theory is.

And so I started with ammonium and then amino-ammonium (which is hydrazine), hydroxyl ammonium, methyl ammonium, fomamidine, amidine, guanadine, methyl guanidine, hydroxyguanidine, aminoguanidine. I just looked up, mostly it was Eastman Organics and Aldrich catalogues. I kind of wrote down, okay, you put another nitrogen next to this nitrogen, what do you get? And I would look up what’s the name of that and look it up in the catalogue. Or I would just thumb through the pages and look for pictures and say, “Oh, that’s an interesting compound.” So I believe that I tested every compound that exists, which is smaller than the sizes that didn’t go through the hole. And so finally I had what I felt was the comprehensive list, and then I had to explain, what does it mean? And that turned out to be pretty hard. The greatest difficulty was that methylammonium did not go through the sodium channel, and yet something like aminoguanidine, which sounds a lot bigger and is sort of a lot bigger, does. So how can you make a hole that does accept the big one and doesn’t accept the little one?

Part 7: Breakthrough Publication
Hille works for a year to formulate his conclusion that the hole was only one of the defining features in selectivity.

Kandel: So that’s how you came to the conclusion that the hole was only one of the defining features in selectivity.

Hille: Well, actually, one struggle that was going on at that time was people had, up until that time, wanted the hydrated ions to be what goes through the hole, and the hydrated ions were a big fuzzy ball surrounded by water. And I was looking at my data, and I thought, you know you couldn’t possibly know what was inside the fuzzy ball at the level of what’s the difference between hydrazine and methyl ammonium. They’re virtually identical, so similar. How could you know? So I thought you just had to take the water off because otherwise you couldn’t see what was in there.

And so I took the water off and made the hole small. And then I realized that if you allowed amino groups, which have hydrogens on them that can hydrogen bond, if you make a hydrogen bond and you buy the CPK models of chemistry and you build them, you see the hydrogen bond. Actually the amino group gets much closer to the group that it hydrogen bonds to than it would if the hydrogen was built with the full round radius of one Angstrom that a hydrogen has. Hydrogen bond means you shorten the distance. So I thought, well, if you make the channel out of material that accepts hydrogen bonds, then those molecules that can make hydrogen bonds will look small, because they can get much closer, whereas a methyl group, which can’t make a hydrogen bond, doesn’t look small anymore. And that was the trick. So I made a hole that was so small that a methyl group couldn’t go through, but anything that could form a hydrogen bond, like an ammonium, looked small so it could pass through, even though if you don’t have a hydrogen bond it looks just the same size as a methyl group. So that was really…

Kandel: That’s remarkable insight.

Hille: Yeah, and it took me a year. I mean, I sat with these data for a whole year while I just sat and wrestled with the problem of, do I have to take all the water off? So here is a case where I just had all this stuff in front of me, the data, and you couldn’t publish because I didn’t know what to make of it. It took a long time just to think about it. And then the first paper that came out promised four more papers—I just read it today—which were papers that came out over the next four years on a variety of subjects that we’ll be getting to shortly. So those two papers, actually, defined the shape of the pore on the basis of criteria that no one had ever used before. And you had no way of knowing whether it was the right answer because you didn’t have anything whose structure was known for which those arguments had been used. So it was a hypothesis that just has sat there. In the following year, I did it for potassium channels as Clay Armstrong and Bezanilla also did. And we both came to the conclusion that it’s about a 3 Angstrom hole on the basis of the same kind of arguments. And that’s what Rod MacKinnon has shown us in his crystal structure of 1998. And so for the first time, we had a calibration that maybe that method does work. I hope it works for sodium channels, too. We don’t know that yet.

Kandel: (Laughter.) We don’t, but very likely it will.

Hille: So that was defining the size and shape of the hole, for which we don’t yet know if the answer is right. We only have this logic. At the same time, I found that a certain ion would go twice as well, or half as well or whatever, as sodium ions by the criterion of measuring the reversal potential, which is the voltage at which the current goes to zero. Let’s say you had sodium ions on both sides, if you replace the sodium ions with a test ion, you now have to go to a different voltage to keep the net current from going outwards or inwards or it doesn’t stay the same. So there’s a criterion for measuring permeability, which is the one I used first, which was on the basis of this voltage as predicted by the Goldman Hodgkin voltage equation. But while I was doing it, it was obvious that the ions that I said were half as permeable as sodium ions often gave only 1/5 of the current or 1/10 of the current. So the size of the current gave a different answer for what the relative permeability of the ion was in comparison to the reversal potential.

And this is getting very technical now, but it was bothersome because in the theories that Hodgkin and his followers had written down, those things should be exactly the same. Those are theories based on what they had called the independence principal. And so I had to wrestle with what was going on. In the first papers, I wrote that the ion acts like an anesthetic. It seems to reduce the number of channels working, while also being a permeable ion. So then I began to think about the ion entering the channel and just sitting there a long time. So if the test ion goes into a channel and sits a long time, if it, let’s say, in competition with sodium ions, goes into the channel just as easily as a sodium ion, but it sits there a long time and doesn’t come out the other side until later, then that ion will certainly carry small current, because it sits in the channel a long time and the channel is tied up. Like having an accident in the tunnel.

On the other hand, from the voltage criterion you could show that this kind of situation where the test ion goes into the channel just as easily as sodium but it stays a long time, it on the voltage criterion would come out to be equally permeable. So then I began to understand how you could get a difference. And to describe that, I decided to use Eyring rate theory, which is the idea that ions jump over barriers and sit in wells and jump over another barrier and sit in a well, as they are proceeding through the channel, not quite like free diffusion. Now Eyring rate theory is something that I had already learned at Yale in biophysics. So in my biophysics major it was one of the things that we did. And at that time I had even read a paper by Walter Woodbury in which he had applied it to axon permeabilities in 1949. So Walter Woodbury has a paper back then.

Kandel: It’s funny, because I always had the feeling that you were one of the earliest people to apply that.

Hille: Well, so there is actually a literature in which Henry Eyring himself said the Eyring rate theory will do for anything, so it will do for diffusion, for example. And Walter Woodbury was probably a post doc in Utah with Henry Eyrring and became interested in nerves, and he wrote an article about that. Now in his article—it’s probably Eyring, Lumry and Woodbury or something like that—in his article, they didn’t have the idea that the ion was staying a long time in the pore. They just used the barrier height as a way to get higher permeability or lower permeability. So it didn’t do the thing that I was trying to describe.

Kandel: Energy barriers of various kinds within the channel.

Part 8: Further Research
Hille writes a paper on the various things that would happen if you had a long pore, as described by Eyring rate theory, in which you allowed multiple ions to be present in the different sites, but they couldn’t go by each other.

Hille: It had energy barriers, but it didn’t have the wells which retained the ions. So the structure was there for thinking about it, but no one had actually applied it the way we did. I wrote a paper on describing sodium channels that way, and for that paper, I assumed that only one ion could be in the channel at a time. So if there’s an ion there, no other ion can go through. And that described what we called deviations from independence—this paradox of two kinds of permeability in sodium channels. And then later, Wolfgang Schwartz, who joined me as a postdoc, and I tackled the harder problem, which is what happens if you have several ions in the pore at once. It’s just harder from the mathematical point of view. Which was something that Hodgkin and Keynes had suggested for the potassium channel—they had some big number of ions.

And so we wrote a paper on, or did a lot of thinking on, that—it took a long time to think about that—and wrote a paper on the various things that would happen if you had a long pore, as described by Eyring rate theory, in which you allowed multiple ions to be present in the different sites, but they couldn’t go by each other. They had to wait for the next one to move out of the site before you could jump into that site. And we described what Hodgkin and Keynes had first described, which was their so-called flux-ratio experiment with isotopes going in and out. That is what led them to this conclusion in the first place. But we went on to show that you could get other phenomena, like if one of the ions was a blocking ion that could go down a certain number of sites, but just couldn’t get over the last barrier, let’s say, it would block the channel if it was pushed down into there, you could get quite unexpected high voltage dependence of that block.

And this we suggested could be used to explain how channels rectify—inward rectifier channels or potassium channels that don’t let potassium go out of the cell, but let potassium ions come into the cell. And if you decided that there was a blocking ion in the cytoplasm that would go into the channel and plug it up and the channel had multiple ions in it at once, then you could get the peculiar phenomena that are known for inward rectifiers. You could make the current size depend upon the concentrations of potassium on the two sides. You could make the block very voltage dependent, and so forth. So that paper used Eyring rate theory again. We sent it to the Journal of General Physiology, and they couldn’t decide about it. It took five reviewers.

Kandel: They didn’t understand it. (Chuckles.)

Hille: Well, no, they could understand it. There was, of course, the debate about whether a theoretical paper deserved to be in the Journal of General Physiology. But there was also the debate about whether the potassium channels really were pores, because even in 1978, which was this time, there are models of inward rectifiers, for example—which are potassium channels—that worked well when they were carriers. And so I think the overall resistance was that, you know, here we were giving sort of unfettered salesmanship to the idea that all potassium channels are pores, acting as if it was something we could just sort of start with when we didn’t even know it. So I think that was one of the last times that I remember encountering complete resistance to the pore theory.

Okay, so I think that covers our sort of physical chemical exercises. There are others into what ions see when they go into the pore and how they feel when they go through it. Now I should say, all of this actually dates back in a way to Clay Armstrong’s first research, which was tetraethylammonium ions block potassium channels by entering from the inside. They go into the channel, they plug it up, and potassium ions can push them out. And these ideas, which he started with in 1965 when I was just beginning my own experiments, are ones that came into my own work from him, all the time… repeatedly.

Kandel: Maybe this would be a good time for you to discuss a little bit about the actual, you know, interactions between the two of you and the influence that might not have been dependent on your talking to each other—or were the two synonymous? Did you talk to each other a great deal or did you primarily influence each other by reading each other’s papers?

Hille: I guess both actually. I looked back at that question also to try to figure out what we had done, and I found a letter from Kenneth Cole to me in 1966. I had sent …

Kandel: Now Clay was Kenneth’s student, was Cole’s student, wasn’t he?

Hille: Clay was at the NIH. Clay was an M.D. And I think at the NIH basically you could do your residency, you know, as a scientist instead of…

Kandel: Yes, I was there also. So you could take a postdoctoral fellowship with somebody there.

Hille: You didn’t have to go be in the war or whatever it was.

Kandel: It was not a substitute residency, it was a substitute for the draft.

Hille: There we go! (Laughter.) You know better than I. So Clay was in Kacy Cole’s lab at the NIH doing this. Also FitzHugh and I think Knox Chandler, Bob Taylor and a few others were there at that time. So I sent my draft of a paper, my first paper on tetraethylammonium ions, which I wrote in ’66 and was published in ’67, I sent it to Kacy Cole. I actually met Kacy Cole in ’65, and it turned out we corresponded almost every month from then on, surprisingly. He was a very active corresponder.

Kandel: For how long?

Hille: Oh, until maybe ’71 or something like that. Lots of back and forth. I have to tell you how I met him. I met him because in the summer of ’65, I worked, in the summer I worked in Harry Grundfest’s lab in Woods Hole, and I did that because I liked Woods Hole. And I asked Alex Mauro, how can I go to Woods Hole? And he said, well, I know Harry, and we can do that. So I interacted with Kacy Cole in Woods Hole, and he was interested in what my conclusions were of that summer’s work, and we talked about it. So I sent him my work on tetraethylammonium ions in ’66, and he wrote back and said, “It’s interesting in this way and here are problems with your grammar and so forth, and Clay Armstrong is doing some work that’s closely related. You should talk about it, and I shouldn’t be the one who tells you about it. Instead why don’t you send your paper in to Clay and maybe he can send you his.”

So I saw a letter there that said, “Dear Dr. Armstrong, I am sending you my manuscript at the suggestion of Dr. Cole.” So that was in 1966. I think that was really the first time—I had read his paper in ’65—but the first time I interacted. And then by ’68, ’69, ’70, Clay and I were corresponding quite regularly, and I reviewed every paper he published for ten years for the Journal of General Physiology, and he probably reviewed mine starting in ’69. So we had lots of interaction. Then in 1970, he wrote me a letter in the summer and said, you know there was a paper in Pfloger’s Archiv from Vogel and Kopenhoffer that said if you cut the ends of a myelinated nerve fiber in tetraethylammonium ion, it will go inside and block from the inside, and it looks different from blocking from the outside. And he proposed that we do some experiments together.

Kandel: You collaborated with him.

Hille: Yes.

Part 9: Collaboration and New Line of Studies
Hille talks about his collaboration with Clay Armstrong and his later work on local anesthetics.

Hille: And so by November of that year, in 1970, he was in my lab and we spent two weeks here doing experiments together. We published together, and it came out in 1972. We explored the idea that tetraethylammonium ion was blocking from the inside, waiting for the gate of the potassium channel to open, and this would show that the gate was on the inside side and so forth, which were the same themes that he was developing in the squid. And then I found letters which said, “Clay, I thought your manuscript was wonderful,” and so forth, where we were trading manuscripts with each other and criticizing them. I think when we saw each other in 1970, we both took care to tell each other that we both thought the other person wasn’t doing interesting experiments anymore and ought to change to some other subject. (Laughter.)

I think I told him basically that TEA is pretty well finished, why don’t you do something else. And I had already published my first paper on selectivity, which Clay was careful to tell me was a pretty weak paper. It wasn’t the good paper, actually, it was a sort of preliminary paper in PNAS that Kenneth Cole had sponsored for me. So anyway, we had an influence right from the beginning. And certainly reading Clay’s paper of 1965 after I had done two months of experiments with TEA, you know that was very influential as an interpretation, it turned out that he was studying TEA that acts from the inside, and I was studying TEA that acted from the outside. Actually one of the reasons he came in 1970 was that he didn’t believe my idea that it was on the outside. And what we ended up finding was there is a receptor on the outside, and there is a receptor on the inside. And so then, everybody was happy with that. (Laughter.)

Another substance that blocks from the inside is local anesthetics. And even in my thesis—as I said the very first experiment I did on the computer with the node of Ranvier was with Lidocaine. It turned out that Alex Mauro, who was my thesis advisor, knew George Camugis, who was in Astra Pharmaceuticals in Massachusetts, and so therefore, we had a supply of Astra compounds of which Lidocaine was one. And we even went up there and talked to them, and they made some more compounds. So for ten years I had a good interaction with the Astra people on getting various derivatives, analogs of local anesthetics, which might test better some concept that I had. So local anesthetics blocked sodium channels. I got a quaternary anesthetic, which would be a charged one that doesn’t go through membranes, and Narahashi had shown that putting those inside the cell blocked sodium channels.

So I gave that to my first postdoc, who was Gary Strichartz, and Gary put this on the inside of node of Ranvier, cutting the ends the way Kopenhoffer and Vogel showed you could let it diffuse to the inside of the node. And then he started telling me that if you stimulated the axon repetitively, the amount of block started to increase. The sodium current would get smaller and smaller and smaller each time you stimulated it. I said, do it again. So he would do it again, and he said same answer. And I said, look, I’ll do the experiment with you and show you how to do it. And it still did that! So that was the origin of use-dependent block, where apparently just like the potassium channel, there’s a gate on the inside, a quaternary ion, in this case the local anesthetic analog, waits until the gate opens and then it can go into the channel.

So that began an interesting many years of experiments with local anesthetics which in the end after working with quaternary ones, I decided I was going to try to figure out again what form of the anesthetic was active in the free amine. Was it the charged form or was it the neutral? So I did a lot of experiments with pH changes, which were to change the ionization of that group. And it’s those experiments which led me finally to what I called the modulated receptor hypothesis. And that idea—which once again is really just an extension of Clay Armstrong’s first idea with potassium channels from his very first papers—the idea was that the reaction of the local anesthetic molecule depends upon the gating state of the channel. If the gate is open, it can go in or out. If the gate is closed, it can’t move. And I added, if the channel is inactivated, which is yet another gating state, the anesthetic binds more strongly. Then I decided that ionizable anesthetics, which have a neutral form, can actually come and go from the channel even when it’s closed… that they can get into the channel through a route which isn’t through the gate, unlike the quaternary ones which are completely trapped.

So I developed the idea that anesthetics—bound to the channel more strongly when it was inactivated—so I developed a model where there were three gating states, the resting channel, the open channel, and the inactivated the channel, and each one had a different affinity for anesthetic. If it was a quaternary anesthetic, it was limited by the gate closing to not moving at that time, and it could only move when the gate’s open. And if it wasn’t quaternary, it would come and go through the membrane into the channel pore, somehow. And I said, the neutral and the charged forms are both active and binding and blocking in the same place. That was the modulated receptor hypothesis. It would lead the channel to become more and more blocked if you stimulated rapidly. That’s because the channel would become inactivated and bind the anesthetic more and more firmly.

Kandel: I just wanted to make sure that you would discuss two implications of that as you are talking about this. One is the fact that, if my memory serves me, this is the first introduction of the inactivated state as being a discrete state that had significance beyond just inactivating the channel—that it affected how ligands bind to the channel. And the second thing is, it indicated that the gating conformational change is quite widespread and affects the whole extent of the molecule, if you will.

Hille: Yeah, okay, and then there’s a third thing that we’ll get to, which is how this relates to antiarrhythmias, antiarrhythmic action.

Part 10: Modulated Receptor Hypothesis
Hille elaborates on the inactivated state, the gating conformational change and antiarrhyhythmic action.

Kandel: Three points of the modulated receptor hypothesis.

Hille: So one point you made was, it brought in the inactivated state as a real state, and this state somehow has a global influence that isn’t just whether the ions can go through, but it does something else. And those same ideas were coming out also from work with scorpion toxins. So it turns out that a peptide, the scorpion toxin, can bind from the outside. This is work that was done in my laboratory actually by Mike Cahalan in ’75. It combines from the outside and affects inactivation, and the amount of binding was affected by what the membrane potential was and whether you depolarized the cell. So there in parallel, all these things were happening sort of at the same time.

Here was a peptide, a big molecule that could bind to the channel and seemed to know on the outside what its state was, and affected this state call inactivation. So there’s kind of another global thing. So within the lab this was now becoming common and easy to accept. But it’s true, the kind of experiments which Clay initiated, that gates affect the binding drugs, added a new dimension to what gates must be, because it wasn’t just whether an ion goes through, it was a whole bunch of other properties that you could now see. And today people tie fluorescent molecules to different parts of the channel and watch the fluorescence change when the gates change. They can see the same thing even better—more beautifully.

Okay, so this work on anesthetics: Throughout, the reason I did it, was because I thought it would tell me the shape and size of the pore. Something about the mechanics, and where the machines are, and what the gizmos are inside the pore. And I was completely oblivious to the fact that cardiologists were using these same compounds to control arrhythmias of the heart and that in arrhythmias, when you had too early or too frequent action potentials, the drugs would work more strongly, which is really the same as the use dependent block by local anesthetics. We published our work without any reference to that concept, and at the very same time Hondeghem and Katzung published a paper which was sort of a theoretical paper about arrhythmias and the action of antiarrhythmics, which are these same kinds of compounds, on sodium channels.

And it turned out, basically, we published the same ideas contemporaneously. And this, the fact that it related to cardiac things, meant that suddenly I was being invited to meetings where either anesthesiologists or cardiologists were involved to describe this work, which was very relevant to them. But I have to confess, we didn’t do it because of them. It was a kind of serendipity of basic science, or our blindness maybe even, coming up with a result which we should have known was interesting to them. But we weren’t doing it because of them, and we didn’t know about them, interestingly. Because it relates to those things, the two papers I published then are actually some of the most highly cited papers I have. I have hundreds of citations.

When we were mentioning the pore size, we didn’t go on to the pore size of the nicotinic acetylcholine receptor, which was later work that comes about now. This is around 1979. And I got the idea that since people were beginning to do biochemistry, and eventually cloning, on channels that it was a good idea for the physiologist to tell the biochemist what the properties of the channels were so that they could look for that. And it would be embarrassing, I thought, if the biochemist told the physiologist what the channels were.

So I said, you know, people like Arthur Karlin and Jean-Pierre Changeux and those people, they’re going to get the nicotinic acetylcholine receptor. We better find out about its pore, because it hasn’t been described well. So Terry Dwyer and David Adams did a whole big series of experiments doing the same thing as I did with sodium channels and potassium channels, looking up every ion—I did the looking up of the ions, they did the tests—every ion that would be of a certain size, see if it goes through. And in this case, we bought and tested 50 ions before we found one that wasn’t going through. And then we tested another 25 or 30 more that didn’t go through, but it’s a big job. They did a lot of work.

Kandel: It was a big hole.

Hille: It was a big hole. It surprised us.

Kandel: Yes. It was astonishing.

Hille: We got another methyl group and another methyl group and another methyl group, and it just kept opening its mouth and saying, sure, I’ll take that. So I had bought already the CPK models, the chemistry models for making model molecules, and I built all the ions as, all the bigger ones. And I took a piece of Styrofoam and addressed it with these molecules. And I cut with a scalpel the hole, and then I would stick another molecule into it, and if it wouldn’t go through the hole, I’d shave away some piece until it would. Then I’d do this with every one of the ions, and finally I had a hole that everybody would go through. And it was about 6 by 6 Angstroms, which is a big hole.

Kandel: Gigantic yes.

Hille: So there’s a case where actually if we are talking about sodium and potassium, they can go through with lots of their waters. Those are the ions that would normally go through. Okay so then, again because of the biochemists coming into this work, I felt it was time to write up for the world, for biochemists and others, what we knew. So this was writing my book.

Kandel: I see, so you had in mind in fact that the field was beginning to be invaded, as it were, by people from other disciplines who needed guidelines to the structural characterization of ion channels.

Hille: Yeah that’s sort of, I mean, I put it in a slightly negative light. There were two things. There were new people coming into the field. Patch clamp was just being invented, so that also attracted a lot of people, and I felt that classical work had determined a great number of things. And I was already finding that the people who were doing biochemistry on the nicotinic receptor—not Arthur Karlin, but others—were discovering, let’s say, desensitization of the receptor and giving it a new name and not knowing that there was such a thing that had a name and that had a model and that Katz and Theslef had tested and had made a nice description of it and so forth. So these were the kinds of motivations that… There were new people coming in and they couldn’t read this literature, actually, which was pretty complicated. It’s difficult to read the Hodgkin, Huxley papers, and it’s certainly difficult to read other papers like even Clay’s and my own.

Part 11: The Book
Hille outlines the process, subject matter and motivations behind his 1984 book.

Kandel: You had written a couple of wonderful reviews. I don’t just mean the handbook one, you had done what, I forgot, the pharmacological reviews?

Hille: Almost every two years, I wrote a review. That’s right. The first one was in 1970 in Progress in Biophysics. Yes. So I had been writing reviews. So now I had about three motivations to tell you, and I had I sort of started in on one, but we got on another, and that is that I had written a lot of reviews, and they had taken a lot of time, and they were kind of long, and they often appeared in books that were published very slowly because they had other authors. And I thought, why don’t I just write a big review by myself. I could even get paid for it, maybe. And I would be in full control of what was in it and when it came out.

Kandel: Absolutely.

Hille: So that was one thing. And I thought, well you know, I’ve actually written a lot of reviews and so those would be the contents of quite a number of chapters already. I could almost take them and render it into the sequence and make my book. And in addition, I had taught an advanced course here in Seattle, where every year for ten weeks I reviewed some literature. One year it was calcium and calcium channels and calcium actions and all that, and I said in 1973, calcium is the ion of the future. And so I already had all the material I needed for a chapter on calcium channels underway. And I had done this for many other subjects, and each one could be a chapter in the book. And many of them were actually not published, because they were just the things I did in classes.

So there was that motivation, and then there was the motivation that we had to make sure that people didn’t rediscover stuff that was already known because it was hard for them to read about it, and so I had to make it more readable. And then I felt that biophysicists were really the people who had been doing all this work, and biophysics had somewhat run its course, by itself. I felt that if we just voltage clamped and could only measure open or closed, all these other states that we postulated or hypothesized, that we were not likely to learn everything you could know about channels, and we needed the help of biochemists, geneticists, pharmacologists, and all the other people.

Kandel: Structural biologists.

Hille: Structural biologists, eventually, fortunately. So I thought, well, you know what I could do is I could write a book where the chapters start very easy, and somebody could read it who was just a biologist. And then the chapters get harder and harder, but they do it so gradually that you don’t even know, you don’t even realize that it’s getting hard. So it won’t be like starting a book that’s hard, that says this field is too hard, I won’t do it. But you just keep going and getting fooled into reading the next chapter, not realizing that it’s getting harder and harder. So that was my strategy.

And I also felt that you had to be able to read the chapters in a finite time, so you had the satisfaction of saying, “I finished that chapter.” So I took every chapter as I wrote it, and I read it out loud. And if it took me more than 60 minutes to read out loud, I would say it’s too long and I’d have to make another chapter. So I actually delivered it orally. Any place that I slowed down while I was reading, I would mark in the margin, come back to and say there’s something wrong there. So those are different things about this book. It finally came out at the end of ’83 and had a copyright of ’84. I think that’s the book. The book went into the second edition in ’92, and now I’m writing the 3rd edition and I hope that it will be available in a year and a half or so.

Kandel: In the context of the book, it would be good if you brought in Rod MacKinnon and the structural biology and how the structure that he ultimately delineated was compatible with the work that you had done and Clay had done, and sort of provide a synthesis of our current understanding of at least potassium channels.

Hille: Okay, so if I say that it confirmed everything we said, I guess that would be kind of a proud, stuck up point of view.

Kandel: But it’s true.

Hille: But it actually was wonderfully reinforcing of what we had been doing.

Kandel: But maybe you could give specific details.

Hille: What Rod showed was that in this channel that he had crystallized, which came from a bacterium and doesn’t have the gates that open and close with a voltage dependence—it may in fact, in fact we know it opens and closes in response to something, but we’re not too sure what the right stimulus is yet. So it may even have some of the gates that we were looking for. The idea that Clay had started and I reinforced through our sodium channel work and our potassium channel work was that the major gate was on the inside, on the cytoplasmic side, and that, when it opened, revealed a big vestibule, which was large enough for large molecules like the local anesthetics and the quaternary tetraethylammonium ions and other things to go into and get stuck in, even when the gate is closed again. So there’s a big space, kind of a closet there where things can be put and the gate will be closed and they stay there.

We could measure the voltage dependence of the entry of these molecules or the number of molecules going into that place. And from my lab we had proposed that the voltage dependence comes because these molecules, which are charged like tetraethylammonium ion or the quaternary anesthetics, are driven by the electric field and pulled into the channel down a certain distance and the electrical distance, how much of the electric field they get pulled through, is easily seen by the voltage dependence of their binding. Now Clay had another interpretation. He said the voltage dependence came from potassium ions that were coming from the outside or the inside and striking the bound molecule and driving it on and off. And actually I think both are correct; they work together, as we showed in our long pore theory paper in ’77. So we had the idea, we found out that molecules would go pretty far from the cytoplasm towards the outside.

That was my view at least, and this means that the narrow place where the selectivity filter—which says this is a sodium ion or this is potassium ion and I like it or I don’t like it—that narrow place must be very far towards the outside of the cell, because these big molecules can go so far into the pore. And then we also, Clay and I and Bezanilla, said the potassium channel has a certain size, a diameter of 3 Angstroms, in order to just sort of fit a potassium ion, not let a cesium ion go through, and it’s coordinated probably by a bunch of oxygens, which make a dipole that it likes as substitutes for the water that it would normally have. And all of these elements were found in Rod MacKinnon’s structure. There was a narrow place, which had oxygens available to coordinate potassium ions. There were, in fact, potassium ions in the crystal, and they were sitting there, coordinated. There were several potassium ions as Hodgkin and Keynes had first proposed.

And then there was an enormous, surprisingly big—I can’t remember what Rod calls it—but there was a big cavity in the center of the membrane, half way through the pore, a water filled cavity in which there was also at least one potassium ion, maybe two. And so all of these things came from that. Rod has recently done theoretical work which argues, I think very nicely, that the cavity in the middle, the water-filled cavity is the trick that overcomes the electrostatic problem that an ion would have as it goes through a low dielectric lipid region of the membrane. If you make the ion go through a narrow pore without water all around it, the dielectric constant is very low and the ion doesn’t like to be there energetically. But if right in the middle of the membrane, where this problem is the very worst, you make a big puddle of water…

Kandel: A lake.

Hille: A lake. Yeah, it’s really quite happy. It’s not really out of water even though it’s right in the middle of the membrane, and then it can proceed through the rate-limiting barrier of the selectivity filter on the outside and all this electrostatic problem is taken care of. And Rod has made very nice calculations actually, with Benoit Roux and others, showing how that electrostatic stabilization works. I think those are really the features of Clay’s work and my work which come out. Subsequent work after ours, before Rod’s, like the work of Gary Yellen, identified residues that were amino acid residues on standard K channels that were facing the cytoplasmic regions, and ones that are facing the pore region…

Part 12: Roderick MacKinnon
Hille shares his impressions of and talks about his professional relationship with Dr. MacKinnon.

Kandel: Yeah, but some of that was done with Rod, right?

Hille: Some of that was done with Rod, yeah, that’s right. But particularly with cysteine scanning mutagenesis and things of that kind, residues were identified that had those properties. And on Rod’s structure, those groups were exactly where you might expect them to be in the crystal with exactly those kinds of positions as described. That was work that Clay and I didn’t do, but again another one of the wonderful confirmations that one gets from the structure.

Kandel: Did you actually interact much with Rod on the course of your…

Hille: Very little really. Rod was a student, a postdoc with Chris Miller, and Chris was a very good spokesperson—not that Rod isn’t a good spokesperson, but I sort of always heard Chris at Gordon Conference, you know, giving the stuff. For the second edition of my book I see that I liked Rod’s work a lot, and there are at least eight papers by Rod that are cited in the book. And I remember I sent chapters of the book around for people to read. I sent several chapters to Bruce Bean, and he pointed out that I had misspelled Rod’s name and he had gone down the hall to confirm, since Rod was there, that I had misspelled his name. So that sort of shows that I hadn’t really in 1991, hadn’t even gotten Rod’s spelling right. In 1991, maybe 1990, I heard him give a talk, and then I began reviewing some papers of his that impressed me tremendously…

Kandel: Yeah, he’s terrific.

Hille: And so around then is when I got to know of Rod and see him mostly at Biophysical Society meetings and the Gordon Conferences, and otherwise we haven’t had a lot of interactions. He’s been here several times to talk. I considered him at those times to be sort of a visionary preacher. He had a vision in his mind, which wasn’t actually fully specified from cause and effect in scientific arguments. He had kind of a conviction about certain things being the case, which he would then present to the audience almost in a mystical way, telling us of how wonderful it was—and he was right normally. I remembered him as a person who had this style—it certainly wasn’t a drab style of speaking.

Kandel: No, he was very engaging. What really sort of impressed me about him was this sort of Babe Ruth gesture of pointing to a set of fields and then hitting a home run, and that is he said, what needs to be done is we need to get the structure, and there’s no use my continuing to do biophysics. I’m just going to quit, find an environment in which I can have the kind of support I need in order to do this structure. And then to do this, despite the fact that a lot of professional structural biologists are trying it.

Hille: I mean here was Rod, who is training actually through Chris Miller, well his training largely was like the things like Clay Armstrong and I did, that is, electrophysiology, using those same kinds of things that we developed. And Chris Miller is in the biochemistry department and more biochemically inclined himself, so all those things probably impressed him.

Kandel: In fact, Chris was trying to get the structures, so this was what they did.

Hille: But that Rod would just throw it all away was really burning the bridges behind him. “I’m going to quit this and do the right thing.” We all knew it was the right thing. Nobody dared to do it. Nobody thought you could just quit if you weren’t already an expert in it and just pick it up and do it. He did it.

Kandel: It was wonderful.

Hille: Yes. So now maybe we should talk about things that we did more recently.

Kandel: Before we get on to that I just want to make sure that we have covered all the topics that you consider important. Up to the modulation, the more recent…

Hille: Yes. I think we have.

Kandel: Okay, good.

Part 13: More Recent Research
Hille describes his experiments demonstrating that a G protein without a diffusable messenger being involved can act on an ion channel and other work.

Hille: Let me say that after writing the first edition of my book, I sort of lost steam in a way. This was 1982, and I looked at what I had written, and there were 16 chapters at the time, and I had done a little bit of every chapter myself except for the Hodgkin Huxley, and I had done research work in those areas, and I had sort of exhausted the things that I felt I could solve in those areas. I think in a way, you work for a while in a field and have maybe a wonderful idea to begin with, and after you’ve worked through that idea, that is the best thing that you have, and you might as well move to a slightly different one, where you might have another wonderful idea. You shouldn’t just stay on the same one. You may not be so inspired, and it might just get monotonous. Now I know you haven’t worked that way. You worked on one idea and carried it forever and made it go in a more and more molecular way, and that’s absolutely fantastic.

But I don’t think that I can do that. I felt that I had sort of exhausted the way I could approach these biophysical questions. And I thought also, more or less as I had felt when I had started at Rockefeller, that actually this very biophysical research was less biological than I wanted to be and somehow was all tied in hypothetical structures and hypothetical states and curve fitting and arguments about that sort of thing. None of which was material and very little of which had to do with how an organism functions in its life and does its thing. So I was looking for other things to do, and that’s where ultimately, after thinking about a variety of things, we came into the G protein coupled receptor modulation of ion channels. And we actually came into that by pure serendipity. It was Paul Pfaffinger, who became a postdoc with you, and in this idea that I had mentioned before that we should tell the biochemists what a channel can do before they tell us.

I said, what’s the next channel that we have to do that hasn’t been cloned yet? And it was inward rectifiers, that very little was known about inward rectifiers. And now we know, for example, they’re blocked by spermidine and other very interesting things that physiologists might have discovered, but it wasn’t until the cloning that it really came out as the thing. So I said to Paul Pfaffinger, who was looking for a thesis problem, how about we look at inward rectifiers. And then I said, now okay, what system are we going to look at inward rectifiers in? So we looked at muscle. We looked at some RBL cells, which have an inward rectifier, and then I said, I know Neil Nathanson down the hall in pharmacology has chicken atrial cells, and we should also look at them just to see what their inward rectifier is before we decide which one you’re going to do this biophysics on.

And Neil was studying the effects of muscarinic agonists on changing the potassium flux in these atria, measured with tracer potassium, he told me, after I went down to his lab and said, can we look at some of these cells? And I said, hey, you know we could do that experiment better. We could do some electricity, and we could see these channels open for you when you add muscarinic agonists. And Neil was interested in G protein coupled receptors, about which I knew nothing. So this collaboration with Neil began with Paul just doing Neil Nathanson’s experiment better. And that was looking for potassium fluxes by looking at channels opening when you added acetylcholine analogs and asking if pertussis toxin blocks it, which was what Neil Nathanson was trying to do with his fluxes. And out of that came eventually the demonstration that a G protein in the pertussis toxin-sensitive Go/Gi family is involved in modulating, opening, the inward rectifier of potassium channels of heart.

Kandel: That was the first direct demonstration that a G protein without a diffusable messenger being involved could act on an ion channel.

Hille: That was the interesting issue. So first we just showed that you needed GTP. We showed that pertussis toxin blocks it, and then there was a literature which said from the past, an experiment done by Soejima and Noma, which was very like the experiment that Steve Seigelbaum had done in your lab, putting a patch pipette on a cell and saying, if you put the agonist in the bath, you don’t get modulation of the channels under the pipette, and if you put the agonist in the pipette, you do get modulation of the channels into the pipette. It’s the opposite from what Steve Siegelbaum found.

Kandel: Right.

Hille: And their conclusion was that there is no second messenger. So this was in the literature already that there was no second messenger. Now Bert Sakmann had been studying this channel, and basically he and Trautwein and Noma, who had been working together, treated this and more or less said it was like a nicotinic acetylcholine receptor that gated very slowly—because you had to put the agonist on the patch that has the channels in it, and you can’t put it somewhere else and have it generate a second messenger that goes to the channel. What we found was that there is a G protein in this process where there’s no second messenger. And I remember sitting at the computer on the very last day that we were going to send this paper to Nature, and Neil Nathason was behind me, and I was sitting at the keyboard, and Paul Pfaffinger was there and we were at this crux, the very last paragraph—what are you going to say? What is the best thing we can say? And I always learned that at this stage you were supposed to say the most bold and wonderful thing that you could say, and that would make the paper probably acceptable for Nature.

And so we debated what we could say. We said there wasn’t a second messenger. We said there was a G protein. And then we said, and then what? The options were to say that the G protein acts directly on the channel, or something lesser, and we chose the lesser route. We didn’t say that it acts directly on the channel. We said, “This is the first time that it has been shown that a G protein acts without using a cyclic nucleotide messenger.” That’s all we wrote. Without a second messenger. We debated both of them, and not having it written on a piece of paper and remembering Hodgkin’s statement, I wouldn’t want to say which side I had of the debate.

Kandel: If you’d been wrong, you would have been very pleased you took that position. So it was an appropriately cautious position.

Hille: If you read our paper carefully, we don’t actually say and we didn’t actually demonstrate that the G protein acts directly on the channel, but we did emphasize that there wasn’t a diffusable second messenger. This was ’85, and in ’87 then, both Neer, Logothetis and Clapham and Birnbaumer and Brown (Buzz Brown) began the arguments about, is it direct action? And then showed that if you put on G protein subunits you get actions. So we get credit of some kind, but we didn’t actually boldly come out and say what we didn’t demonstrate, but what we should have said.

Kandel: You set up the problem.

Hille: We did. Interestingly, then I told Paul Pfaffinger okay, now for your thesis—this wasn’t his thesis, this was a warmup—so what are we going to do? So we switched to another potassium channel, which also used muscarinic agonits, and that was the M current that Paul Adams and David Brown had discovered. And we thought, okay, this would just take a few minutes, and then we’ll have the answer to the M current because it will be the same. And it definitely wasn’t and even today, our laboratory is still studying what second messenger mediates this process, and we don’t know the answer.

And then we started working on calcium channels, whose opening is depressed by G proteins instead of their being opened by G proteins or by receptor action. So as Cathy Dunlap and Gerry Fischbach showed, the current carried by certain calcium channels can be decreased by many agonists, norepinephrine, acetylcholine being among them. And they proposed correctly that this might be an explanation for pre-synaptic inhibition. And we began to study that and characterized it in many ways. Only recently, or maybe ’95 I guess, we showed together with Bill Catterall’s lab that the G protein beta gamma subunits act directly on the channel in that case too. So we sort of came back to this direct G protein interaction. The potassium channel, I guess, was the first case where the beta gamma subunits…

Kandel: Yes, that was the other interesting thing to come out of it, and that is that the Clapham and Neer argument was a correct one, and it was the first demonstration of a beta gamma subunit acting as an effector.

Hille: Yeah, and it was a clear one. And the calcium channel becomes another and there are now quite a number of effectors. Probably more effectors for the G protein beta gamma subunits than the alpha subunit are presently known. Okay, well that’s really the kind of problems that we’re still working on today. How do you modulate ion channels? We’ve also worked a lot on calcium dynamics and other cell biological questions, which probably we shouldn’t get into and aren’t related to the Lasker Award part. I think we’re done, Eric.

Part 14: Perspective On Ion Channels Field
Hille talks about how the field of ion channels has changed from its early beginnings to today.

Kandel: I think that’s wonderful. Give me just sort of a few perspectives on your career, of how the field of ion channels has changed from our early conception to now.

Hille: Okay, changes, there are several. One is, at that time people didn’t know they existed. They didn’t know there were pores and so forth. Every time you began to lecture about doing the kinds of experiments that we did you’d have to spend ten minutes telling people that there is a sodium potassium pump and you’re not talking about that kind of molecule that uses ATP, you’re talking about a different molecule. Nobody knew about it. In fact, nobody cared about it.

So when I began my thesis work, there were fewer than 20 papers to read altogether on voltage clamping, and probably three or four papers appeared each year. And now if you search on Medline, 5,000 papers appear every year, and you can publish it in Nature and Science and Cell and Neuron. It’s a very highly competitive field in which the molecules, which we weren’t even aware of at the time, have been cloned, and we have the proteins. We’re beginning to get the crystal structure of the first ones. We have molecular diseases so that quite a number of human inherited diseases have been shown to have mutations in ion channels of which the most famous is CFTR, cystic fibrosis transmembrane regulator, but many in sodium channels and calcium channels and potassium channels. So the field has grown by several orders of magnitude.

In 1980, Neher and Sakmann and their colleagues published the patch clamp method, and this changed completely the approach. You no longer had to stick wires inside cells and so forth, and furthermore Fred Sigworth and Erwin Neher designed an amplifier, which then went on the market. And this was the first time that you could buy equipment off the rack that allowed you to do experiments with voltage clamps. And since then, about 20,000 of those boxes have been sold by the various groups that now supply them, all of whom designed them because of some electrophysiologist who made a design, and then it was licensed.

I don’t think anybody has designed a patch clamp who wasn’t already a practicing electrophysiologist. So instead of having to make your own amplifiers which I did myself at that time, and make your own computer interfaces, in fact not use a computer at all for most people, now you buy the computer program. You can buy the computer for a very low cost and you buy the amplifier and you buy the stimulator. So basically you just ask for $50,000, and you get a set up completely made, and nobody who works with you has to know how it works. You get on the phone and a service person can guide you through whether you’re supposed to send it back or not. So that is sort of the mechanical side.

Intellectually, I think the quality of work has been high all the time. It’s very high now and it was high from the beginning. Certainly Hodgkin and Huxley take full responsibility for setting the level where the bar is. It’s very high. You have to be good at quantitative thinking and good at logic and so, although we have lots more people, the quality has stayed about the same. But the kinds of things you can do is enormously expanded. Instead of just looking at electrical currents you can see fluorescence changes of molecules. You can test individual amino acids. You can test protein-protein interactions all these things make it just wonderful. So I would say it’s very fulfilling to see the growth of interest. The fact that we had to previously kind of push uphill to even get the audience to open their eyes, and now often people assume that it must be interesting, so they come with bright faces and open eyes and listen.

Kandel: Well, it’s the central theme of neurobiology, and what I really have admired about your work and Clay’s is that essentially you took what was a black box approach. I mean, without seeing the structure you were able to characterize it so satisfactorily that it provided an excellent guide for the cloners and the structural biology.

Hille: I guess it’s really the power of biophysics.

Kandel: That’s right the power of biophysics.

Hille: Or of voltage clamps.

Kandel: And good thinking about it. Yeah. I don’t think it’s just the voltage clamp, and I don’t think the methodology, because you guys really didn’t introduce that much methodologically in terms of modifying the voltage clamp.

Hille: Yes, that’s right.

Kandel: I mean you used the transistorized model.

Hille: And think of how much Hodgkin and Huxley found out just from one set of papers.

Kandel: That’s right. Just a few squid giant axons, as Zeki would say.

Hille: They set the stage, and Clay and I continued with a much more molecular desire.

Kandel: That’s right. That’s what you introduced, the molecular feel of it.

Hille: Though we always had in mind that we were going to figure out what the molecule looked like. Very interesting to me and I don’t have any explanation, that once we could figure out what the molecule looked like at the point of view of cloning, I stopped doing the work.

Kandel: Well, you had solved what was for you the important aspects of the problem.

Hille: Well, I solved what I felt I was good at solving. And so I went to something else.

Kandel: Modulation in the mind.

Hille: There we go. I feel good.

Kandel: I feel very good. I feel you have done a wonderful job in describing your work, and I think it’s terrific work, which should give you a lot of satisfaction.

Hille: Thank you for your generosity in taking us through this.

Kandel: Oh, this is lots of fun. This is very exciting for me.

Key Publications by Bertil Hille

Hille, B. (1967) The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ion. J. Gen. Physiol. 50: 1287–1302.

Hille, B. (1971) The permeability of the sodium channel to organic cations in myelinated nerve. J. Gen. Physiol. 58: 599–619.

Hille, B. (1977) Local anesthetics: Hydrophilic and hydrophobic pathways for the drug-receptor reaction. J. Gen. Physiol. 69: 497–515.

Dwyer, T.M., Adams, D.J., and Hille, B. (1980) The permeability of endplate channel to organic cations in frog muscle. J. Gen. Physiol. 75: 469–492.

Pfaffinger, P.J., Martin, J.M., Hunter, D.D., Nathanson, N.M., and Hille, B. (1985) GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature 317: 536–538.

Herlitze, S., Garcia, D.E., Mackie, K., Hille, B., Scheuer, T., and Catterall, W.A. (1996) Modulation of Ca 2+ channels by G protein beta-gamma subunits. Nature 380: 258–262.