Peter Agre (2014) - Aquaporin Water Channels – From Atomic Structure to Malaria

Guten Morgen (Good Morning)- I didn't hear that. Guten Morgen. It's great to be here. This is I think my eighth trip to Lindau, I always love to come here. It's such a pleasant event. Meeting the young scientists is a great joy and we have a lot of people to thank. In my case a personal visit from Countess Bettina and her mother the late Countess Sonja made it obvious that this is something very important for every laureate. And the organisers, the three Wolfgang’s and their team, as well as the staff here in Lindau, Susanne, Katja and their team, make it such a delight to be here. So I'm going to talk to you about water channels. Water, we take it for granted often. It's essential for life, for commerce, for recreation. There’s something about water that can cause great harm when it's at disequilibrium; can cause hypothermia or drowning. There’s something about water that’s very fundamental to life. I think it was Albert Szent-György who said Water is the solvent of life. In the story I'm going to tell you about has a lot to do with young scientists. Does anybody have a clue who this young guy in the middle is? Yes, I'm sorry the ravages of time are cruel. But all of us laureates started off as young people like yourselves when the passion, the opportunity, the decisions were made often to leave our homes, our families, to strike out and do some scientific exploration with no guarantee anything would succeed. But yet science continues to move on because scientists make discoveries. Sometimes discoveries they expected to make and more often discoveries that were a total surprise. So the discovery of Aquaporin-1, the first molecular channel was pure serendipity. I often tell journalists we didn’t discover Aquaporin-1, it discovered us. It was in fact a protein that co-purified with our subject of interest the rhesus blood group antigen. But there were some curiosities: it was a slightly smaller size; it turned out to have immunological lack of cross-reactivity of RH. And so we did what scientists do. On our hunch that it might be interesting, we purified it, we sequenced it, we cloned it, thinking ah! We can go to the genomics database, it will tell us exactly what this new protein does because it was very abundant in red cells. But it curiously didn’t stain with the protein stains. No one had seen it before. Imagine that, a protein and red cell studied by hundreds and hundreds of scientific groups, a protein that’s present, that no one had seen before because it simply didn’t stain. So we cloned it out and the deduced sequence provided a 6-membrane-spanning domain shown in the slide. So the protein lives mostly between the leaflets of the bio-layer and has an internal repeat: the first half and the second half are genetically related but in an adverse confirmation - predicted. When we went to the genetics database in fact there were some homologs present. There was a homologue for lens of eye from beef cattle - referred to as major intrinsic protein, function unknown. A protein from the brains of insects, drosophila - important in neurogenesis but biophysical function unknown. So proteins from bacteria which seem to be involved in glycerol update, and a series of fragments of genes from plants - again function unknown. So we had a new protein, we purified it, we had a sequence we cloned it. And we did not have a clue what it did. Let me emphasise that: we did not have a clue what it did. And a protein without a function is a scientist without a grant, without a future. You go to your professor - well I was the professor, a young professor in my thirties. So I talked to my colleagues, many of them: a red cell protein must be a channel they assured me. The haematologist: There’s no red cell channel, you are wrong. And so we were stuck. Now this illustrates an important concept, work-life balance. When I was a student, 45 years ago, they didn’t talk about work-life balance, it was all work. If you don’t work hard enough, you will not succeed. But there’s something special that we all have in terms of our friends and our families. So my wife and I have 4 wonderful children, this is a picture of them when they were small and cute, this is 25 years ago, 22 years ago, adorable kids. We took them camping every year in the national parks And as a scientist on a low salary it was the vacation we could afford. So my wife said, you know we should ask the children which national park they would like to go next year. So we did, children which national park you want to go to? They said: Disney World! Well you know, Disney World is actually not a national park (laughter). But families are not real democracies either. So we went to the Everglades (laughter) got bitten by mosquitoes and visited Disney World where the kids had a great time. And coming back to Baltimore - it's a long drive, about 2,000 kilometres back – we stopped in Chapel Hill, North Carolina, the University of North Carolina where in my younger days I was a postdoctoral fellow with John Parker, a wonderful haematologist a colleague, who was a good protein physiologist. And I told him as I told others about this new protein in red cells, we had not a clue what it did. John thought and he said, red cells, where else? I said, renal tubules. Red cells, renal tubules. And it has some structural homologies with proteins from the roots and tissues of plants. And it was John that leaned forward and said, Peter, have you considered this might be the long sought water channels, something that physiologists have been searching for a hundred years to explain how osmosis can occur so rapidly in some tissues. I was astonished - water channels? I never heard of a water channel. But following John’s suggestion, when I returned to Johns Hopkins we teamed up with Bill Guggino to do some studies I’ll tell you about. So a little background here, water is almost ubiquitous in biology. But how does water go in the directions it needs to go for proper homeostasis? How is it that all of us can be secreting and releasing and resorbing cerebral spinal fluids to bathe the surface of our brains? To fill the orbits of our eyes with aqueous humour but not too much? To salivate, to sweat, to humidify our airways, how can the roots of plants take water up from the ground? And it was probably this concept of water channels that was missing to explain that. So membranes, all membranes, have a finite water permeability and if you look at the left panel you see the fusion of water through a lipid bilayer, a random event with a high Arrhenius activation energy. And it was the pioneers, let me stress, the pioneers, people like Arthur Solomon, Alan Finkelstein, Guillermo Whittembury in Venezuela, Jean Poyet in France, Mario Parisi in Argentina, Gheorge Benga in Romania, these pioneers had done very fundamental work, very sensitive work that indicated there must be water channels. It was this that John was referring to. And the fundamental difference is a little subtle but in fact all biological membranes exhibit some bilayer diffusion permeability but special membranes such as renal tubules, secretory glands, and red cells were presumed to have aqueous channels, water channels. The biophysical differences were again a bit subtle: low capacity diffusion, bi-directional water goes into and out of cells randomly. But those with water channels can have a high capacity for water and selective for water. H2O moves with almost no resistance in the protons in the form of hydronium ions. The same mass with the charge fails to move perceptively. And the direction for the water movement is created by osmotic radiance; so this concept of osmosis that all of us learnt about in school from our teachers when we were quite young had a molecular explanation. Now in 1970, the year I had the backpack and was hitch-hiking through Asia on my way to Johns Hopkins, an experiment was undertaken at the University of California, Berkeley by another fellow from Minnesota, Bob Macey, who went through the chemical catalogue basically and was looking for agents which would create increased water diffusion. So he was hoping with the proper chemical applications that all membranes would have increased diffusion. He came to mercuric chloride and the surprise was, it stopped water transport. Stopped it! And by washing away the mercuric chloride and treating with chemical reducing agents he could restore it, he could turn it off and on like a faucet. When you do this correctly there must be an aqueous pore, the free self-hydro, somewhere in this channel. But neither Bob Macey nor the others were able to identify the channel and that’s where science moves on. We are like a relay race carrying the baton. It was handed to us just at the right moment. So with Bill Guggino at Johns Hopkins we followed John Parker's suggestion and tested the function of this new protein by expressing the complementary RNA in xenopos laevis oocytes - these are frogs eggs. Frog eggs are about a millimetre in diameter. And as we know frogs lay eggs in fresh water ponds in the spring unless they are fertilised they are dormant because they have very low inherent membrane water permeability, an ideal expression system. So the key is: control-oocyte injected with buffer alone and a test-oocyte and injected with 2 nanograms of the complementary RNA; culture three days and no obvious difference. But the culture medium now is isotonic, there’s no osmotic radiant. So if you were to take these cells and drop them into distilled water, the control-oocyte should swell negligibly, the test should swell and explode, which is exactly what happened: like popcorn, 6 controlled oocytes And this produced jubilation (laughter), almost like a Mozart Opera! This is Greg Preston, the young scientist in my lab who did those experiments, and I took this picture of Greg. Something about discovery and science - that’s the energy, the reward. Now, what we were not prepared for was the interest of other scientists, because we had a very small group, very focused: a clinical medical doctor doing research, and there were some large groups who had great investments to discover the water channel. So we had to work in collaboration with others. So I'm going to tell you about other scientists with whom we collaborated because science is a worldwide network of individuals like ourselves. Many of them young scientists and I have pictures of quite a few. So our first challenge was to establish the structure of the water channel protein. The famous architect Louis Sullivan who designed the first tall office buildings, he was the mentor of Frank Lloyd Wright. Sullivan is remembered for having said, ‘Form always follows function’. So if we could establish the form, the structure, we could understand the function of the water channels. And in collaboration with Yoshinori Fujiyoshi and Andreas Engel, I just showed their pictures, we solved the structure at an atomic level resolution. And in the left panel you should see the channel from above the cell. It's a tetramer but each subunit has a single aqueous pore, diameter 3 angstroms. What is the average diameter of a water molecule? No chemists here - Astrid, we have to work on them! And in the right panel you see the aqueous pore extending from the surface of the cell to the cytoplasmic membrane; a single pore along water to move in single file and this diagram indicates how it works. So we call this the hourglass. The ancient timepiece with an internal symmetry, with an extracellular vestibule, intercellular vestibule, where water is in bulk solution, H2O to the nth power, protons hopping freely amongst these water molecules. And then the channel: A 20 angstroms span, about 3 angstroms in diameter, where water moves in single file. We thought we knew a lot about this, there are barriers to the movement, charged solutes and protons, for example an arginine here, a histidine here. And there are other things that go into the barrier for the proton movement. Arieh Warshel, I think on Monday, talked about this. So science moves on but the basic workings I think are pretty clear. There’s a single water molecule hung up in the centre of the channel by interaction to these 2 cytogens, these 2 amino acids, which are highly conserved. So water moves with almost no resistance - and again this is work of David Kozono, who was a young student when he did this work, he’s now a Harvard faculty member. And as you can see water, a single molecule filling the pore. So here charged residues preventing proton movement, assisting the mercury inhibitory site. And the interesting thing about this structure is that it's shared amongst all life forms. So we concerned ourselves mostly with the mammalian, the human aquaporins, of which there are 12. But if you go to the genome, there’ll be at least 500 aquaporin from different life forms with the same basic sequence and the same structure So the human repertoire is split into two subsets: water selector channel, the aquaporin’s, and AQP1 is what we concerned ourselves with most, but I’ll tell you what's in the others. And those that are genetically slightly different permeated by water plus glycerol, the aquaglyceroporins. And most life forms have some of each. So again: how does a small laboratory compete in a big scientific field by collaborating? And this is our scientific collaborator for pinpoint localisation of the aquaporins in mammalian tissues. This is Søren Nielsen from the University of Aarhus in Denmark. There’s only one of you from Denmark here, we need a little more Danish participation here in Lindau. Søren was 29 years old when he did the studies which established the physiological function for the aquaporins. And he did so with classic immunohistochemical and immunological electron microscopic techniques. So the physiologists were keenly aware that our kidneys, we have two of them, each are formed of about a million nephron units, where plasma is filtered in the glomerulus and the water is reabsorbed in the proximal descending a limb, and this is exactly where AQP1 resides. The ascending thin limb then has no water transport. And the collecting ducts have regulated water transport, but there’s no AQP1 in this location. So looking at a cross section of kidney at the apical surface, the water absorbed the surface, the black dots represent immunible decoration. Intense expression of aquaporin-1 protein. So this is the apical surface and notice the cell body has little staining. So it's poised at the surface. It's also present not only in the apical surface but in the lateral and basis surfaces. So water moves from the primary urine and we filter about 200l of plasma - to filter 200l is enormous! and out through the lateral and basal surfaces, with a direction graded by osmotic radiance created by salt and water and sugar transporters. So, as a medical doctor I was very interested, what are the human consequences? And with Landon King and others we identified humans with knockout mutations, and that was made possible because there was a blood group antigen, a polymorphism, on the surface of AQP1. And in short individuals with knock-out mutations in AQP1, as this lady from the south of France who voluntarily lets us show her photograph, they do pretty well. They feel well. There’s a blood group transfusion difficulty, but without needing a transfusion you may not even know you have a defect; but, when stressed, the consequences are quite apparent. So with overnight thirsting all of us would concentrate our urine to about 1,000 milliosmoles, the same concentration as seawater. These individuals with overnight thirsting can concentrate a little bit from physiological saline 280 to about 420 milliosmoles. Enough to get through the night but they can’t concentrate with prolonged thirsting and fusion of hypertonic sodium chloride. So they are stuck. So they have a mild form of nephrogenic diabetes insipidus. Of course with modern life and free access to liquids and air conditioning we are not stressed, but if we were it would become apparent. The second member of the family - and sort of a land rush of interest in the aquaporin’s emerged when we reported the first member - the second member AQP2 is present in the collecting ducts where regulated water transport is known to occur. Aboriginal people are unable to measure time in intervals shorter than a day. But they can time their war parties in the middle of the night because as the Plains Indians would do at sundown, they would drink a large volume of water and a couple of hours later they’d get up to empty their bladders, because regulated water transport is what explains this. Any graduate student who has gone to a TGIF on Friday and drinks a litre of beer knows that the first place he goes afterwards is to the toilet. Playing tennis in the hot sun, the opposite: concentration of urine. So the final volume in the collecting duct is created by AQP2. And it's regulated by exocytosis of intercellular AQP2 containing vessels to the surface and back, regulated by vasopressin. So we have now in the principle cells of the collecting duct an intercellular aquaporin that moves to the surface and back, allowing water to enter the surface, but it exits through another member of the family. And inherited defects are quite important. The children of mutations of the gene expressing this must drink 20l of water a day to avoid dehydration. And acquired defects are common. Fluid retention, such as congestive heart failure, AQP2 is up-regulated, problems such as bed-wetting it's underexpressed. So it's a pretty simple answer to a long-standing physiological challenge. Now I'm going to rapidly step through other members of the family because each one is expressed in a different tissue and had a different group of young scientists. Masato Yasui, who is now a Chair Pharmacology at Keio University, worked on the AQP0 protein in lens and defects caused cataracts in small children. Another member of the family AQP4 is expressed in several tissues including brain where it is present in the astroglial end-feet. They look like suction cups surrounding brain capillaries, because the brain fluid movements are very, very carefully regulated. A closed head injury leading to brain swelling causes compression of adjacent brain tissue and permanent brain damage. The AQP4 right at the perivascular membrane explains the fluid movements into and out of brain. This was established in collaboration with our colleagues in Norway including Mahmood Amiry-Moghaddam, a refugee from a refugee camp in Pakistan. His family had fled Iran at the time of the Mullahs. And he was brought to Norway as a high-school student and he wanted to do science and that opportunity made him one of Europe’s leading neuroscientists. There are 2 of you from Iran at this meeting; we need more participation from all of these countries that have fewer or no representation. So Mahmood showed us that normal brain after defining brain injury sustains significant brain damage. But mice with mutations in the scaffolding protein so the aquaporin-2 is mis-localised have a survival advantage as you can see. The pink represents viable brain tissue after sustaining a brain injury and they do better. AQP5 from saliva glands, lacrimal glands, sweat glands cloned by Surabhi Raina, a young scientist from India. This explains how we can release sweat to cool ourselves when we are thermally stressed. And the aquaglyceroporins are also important. Now this slide I borrowed from Bing Jap from the Lawrence Berkeley Lab in which he has aligned water channel residues in the pore aligning domains, and they are shaded darkly, and the corresponding residues from glycerol transporters from bacteria GlpF, and they are shaded lightly. So this is considered... here’s the cysteine and the water channel, and notice it's replaced by a phenylalanine in the glycerol channel, a larger hydrophobic residue. Here’s the histidine, a partial positive charge in the water channel, and notice it's replaced by a glycine, a tiny amino acid in the glycerol channel. So in the movement of water through water channels this is the aqueous pore, 3 angstroms in diameter. But in the movement of glycerol this hydrophobic back door, like a trap door to open, allowing the movement of the larger 3-carbon polyglycerol. It's amazing how nature adapts a single sequence to do these feats. Now AQP3, aquaglyceroporin 3, is present in a number of tissues including basal levels of skin, where it's present both before birth and after birth. Now a few years ago I was visited by some executives from the Christian Dior Company, and I was a little suspicious. We never had any interest from Christian Dior or any other beauty companies. But they wanted me to come to Paris to give a lecture, to announce the release of a beauty product which is called hydro action skin cream. I should point out I have no financial ties to Christian Dior. So I can speak freely. So this is a naturally occurring small molecule which leads to a subtle induction of aquaglyceroporin 3 in skin. And in the beauty industry you don’t really have to prove your product, and the notion that if you use enough of this, you’ll look like this - well I'm not personally convinced but let the buyer beware. Now this is the back cover of the Marie Claire beauty magazine, those of you who read French will see the statements are a little bold. It says: profound hydration, spectacular results, the Nobel prize in Chemistry! I showed this to my mother, she’s in her 80s, she’s a farm girl, she never went to university. She looked at that, she smiled and said, Peter I think you finally doing something useful! And I think she was kidding, I'm not sure. But I think there is some utility to the studies. Because aquaglyceroporin 3 is also present in red cells. Glycerol transport into red cells turns out to be very important in the setting of malaria. Malaria is my new adventure. Because when a malaria parasite invades a red cell it lives within a single cell vacuole, it can rapidly grow by synthesising glycerol lipids. The glycerol must come across 3 membranes: the plasma membrane of the red cell, the vacuole membrane and the parasites own membrane. In a series of knockout studies we confirmed that glycerol transport was essential for full variance of the malaria parasite. So notice here, a single red, these are red cells, a single parasite divides: 2, again: 4, again: 8, 16, 32. And so in just a couple of days you go from a single parasite to 32 daughter cells. A ... (inaudible 26:15) which breaks releasing 32 daughter cells. And do the numbers: 32 times 32 is about 1,000, and 32 times 32 is a million. So in 6 cycles we have a billion parasites with grams of malaria sludge through the circulations of the patients with horrific fevers. And I’ll skip through this briefly but basically the aquaglyceroporin 3 provide the mechanism for the parasite to rapidly kill Not long enough to make this, I think, a good drug target but that’s research - you try it. So let me get back to the clinical topic of malaria. Who gets malaria? It's the children in the developing world, particularly in sub-Saharan Africa. Little children like these youngsters playing in the villages outside our research station. And I now am privileged to spend about a third of my year in sub-Saharan Africa. I came on Sunday from Malawi where we are working to prevent and develop better pre-treatments for malaria. So these kids are lucky, if they have malaria they are well treated and they are completely normal. But this little boy came in near death to the village hospital where the doctor on call, Dr Philip Tuma, a paediatrician from Johns Hopkins, saved his life. He was comatose, near death, but notice he has a dysconjugate gaze, he’s looking upward because the brain damage from cerebral malaria has killed his optical cortex, his visual cortex. He’s totally blind, he’ll never recover. In many cases malaria will kill children, 700,000 a year but many millions of cases they recover but not totally. So it's still a big challenge. This is a study from mice but if we look at the brain cortex from the child we see that in a normal situation the astroglial endfeet are lined along the capillary basement membrane. The black dots represent immunoglobin staining. And in the case of cerebral malaria the swelling, these blisters of fluid and coagulation, infarct, leads to irreversible brain damage - a big challenge. And of course malaria is spread by mosquitoes, the Anopheles in particular. And the Anopheles have 8 different aquaporin’s, I won’t go through it, but to fly after taking a blood meal they jettison fluids out through the malpighian tubules; their water balance is maintained by aquaporins. And of course mosquitoes cross borders, so any country that controls malaria is protective but not totally. This is the colonial bridge separating Zimbabwe and Zambia. And borders are often areas where malaria is out of control. The border of Northern Zambia with Congo has terrible malaria because the parasites and mosquitoes go back and forth. So in closing I’ll just mention aquagylceroporin 7 in adipose tissue, release of glycerol, uptake through aquaglyceroporin 9 into the liver allows us to maintain blood sugar levels during fasting and starvation - and this is work by Jen Carbrey. But a big surprise, the aquaglyceroporins are also freely permeated by arsenate, explaining the toxicity of arsenic in ground water in Bangladesh, in eastern India. The prevention of this disease of course is the provision of pure drinking water, a basic human right. And plants, this is a slide from Ralf Kaldenhoff when I was here in Germany at the University of Würzburg. The rice genome has 50 different aquaporins and you see here the plant of the Arabidopsis maintains nice foliage. And an engineered form has similar foliage but with the knockdown in the root with aquaporins, the plant compensates by sending out more rootlets to maintain stem balance. So I’ve talked about several topics, I’ll skip briefly to the end. They are expressed throughout nature. And let me tell you briefly about a very pleasant telephone call I received in October 2013. This is an important telephone call from Sweden for Dr Peter Agre, are you Dr Agre? I sure am and I was told I would share the Nobel Prize in chemistry. And they told me they would be having a press conference in 10 minutes and I should get ready for my day. So I sprinted to the shower and my wife Mary called my mother back in Minnesota, again a farm girl; my Dad who was a chemistry professor had died some years before. And Mary told my mother that I would share the Nobel Prize in chemistry and there was a pause, and my mother came on, she said, Mary, tell Peter that’s very nice but don’t let this go to his head, (laughter) he still needs to do something useful. So here we are with the lab group, these are the young people at the day of the Nobel announcement and they came from 8 different countries, a youthful group representing large sections of the world. And here we are in this... oops I forget here - you can’t actually limit the celebration. This is the cut-rate liquor store in the neighbourhood near where we live, where the price of Heineken is usually announced but those days they had a different announcement. We don’t own science, we do science; science is for the public. And here we are on the stage in Stockholm and any resemblance I may have had to Alfred Nobel came and disappeared when I took off the moustache. And here I am with my family. And let me just challenge you young people to go for it in science, the rewards may not be financial but they’ll be terribly important, both for your career and for the world; and don’t forget to thank the people who support you during this. With that let me thank you for attending my lecture and I look forward to seeing many of you at the break. Applause