Waterway to Stockholm

A tear-jerker movie. A mouth-watering aroma. The human body’s ability to suddenly release water is

so commonplace that few of us give it a second thought. But to biochemists, physiologists and others studying the movement of molecules into and out of cells, the phenomenon presented a first-class stumper for generations. Diffusion, a kind of slow-motion seepage through the cell membrane, offered one explanation. Yet it couldn’t account for how quickly our eyes can well up, for example, or the gallons of water our kidneys handle every day.

Some researchers suspected that ion channels -- the proteins that make a cellular doorway for calcium, potassium, sodium and chloride -- did double duty by also acting as a water sluice. Others said, no, water must have its own, specialized gate. But how do you find such a thing? In cells that are already more than two-thirds water, where do you fish for a water channel?

In the 1980s, Peter Agre had heard of neither the debate nor the quest. He was, however, well steeped in the unexpected twists a line of scientific inquiry can take. A 1974 Hopkins medical school graduate and board-certified internist who’d trained in hematology and oncology, Agre (pronounced ogg-ray) had been examining the structural proteins of red blood cells when he encountered the case of two sisters with a debilitating anemia. He not only showed that their hereditary spherocytosis—a rare disease of misshapen blood cells—was a previously unknown form caused by a recessive gene, he found at the molecular level that the culprit was an insufficient amount of the very protein he’d been studying.

And so, when Agre embarked on his next research project, he already had the prepared mind that chance was about to favor. This time he set out to study the substance in the blood that can sometimes cause maternal blood to attack the fetus. No doubt some scientists would have ignored the gooey molecule that kept fouling Agre’s solutions of purified Rh protein. But he followed the urge that said, Take a closer look. What he found was the long-sought water channel. And it was that discovery that brought Peter Agre at the age of 54 the 2003 Nobel Prize in Chemistry.

Walking along the hallway of Agre’s lab on the fourth floor of Hopkins’ Preclinical Teaching Building, you can’t help but notice the images of Native Americans that adorn the walls. Born and raised amid southern Minnesota farmland, in what he calls “an idyllic little Norwegian town,” Agre and his five siblings are 100 percent descended from the low-key Scandinavian settlers who began populating the area after the Civil War. “If I fail to make my grants, I plan to write for Garrison Keillor,” Agre jokes, referring to the author whose humorous tales of life in the Minnesota town of Lake Wobegon have garnered a huge following. (He told a roomful of reporters that his mother cautioned him, “Don’t let it go to your head,” when she heard he’d won the Nobel.) As a boy, Agre remembers sitting on a hillside and waving the Norwegian flag when the King of Norway visited. But he also became intrigued by Minnesota’s other residents—the American Indians who lived nearby. “I would go to pow-wows,” he says, “and I spent hours and hours making a re-creation of an ancient Sioux belt. There’s an obsessive-compulsive side when I get fascinated with something.”

Agre credits his father, who died in 1995, with sparking his fascination for science. Courtland Agre worked his way through college during the Depression by selling shoes at J.C. Penney. He was a professor of organic chemistry, first at St. Olaf College and later at Augsburg College in Minneapolis. “Dad would invite us kids to his lab and have us do little experiments,” says Agre. “We were very proud of him. In third grade, the teacher asked us to draw a picture of what we wanted to do when we grew up. I drew a chemist with test tubes.”

Agre never really abandoned that dream, though at one point he toyed with the idea of becoming ajournalist. He earned a B.A. in chemistry, with honors, at Augsburg, then decided to go to medical school. “I wasn’t confident that I had the ability to cut it in basic science,” he says. “I wasn’t an exceptional student. I was diligent. But as a medical student at Hopkins, the lights really went on. If I had gone anywhere else, I would have gone in a different direction. Here, I met people who were so interested in science that it made me think it would be fun.”

After earning his M.D. in 1974, Agre stayed on for a year as a pharmacology fellow, headed to Cleveland for his residency in medicine at Case Western Reserve University, then opted for a second postdoctoral fellowship at the University of North Carolina at Chapel Hill, where he quickly was promoted to clinical assistant professor of medicine. But the lure of the lab never deserted him, and when Hopkins offered him a dual appointment in the departments of Medicine and Cell Biology and Anatomy, he headed straight back to Baltimore.

It was a colleague, from Agre’s days at UNC, “a superb basic physiologist,” who suggested to him that the gooey contaminant that kept disrupting his Rh study might be the elusive water channel. At first, Agre had thought it might actually be the substance he’d been studying—the antigen that made the blood in some women attack their unborn child. But a bit of delving proved the molecule to be a protein in its own right—and one that was widely expressed. Agre came upon the protein not only on the membranes of red blood cells but in kidney cells and blood vessels, and related proteins in tear ducts, salivary glands, even plant cells. The sheer abundance of the stuff tugged at his curiosity. More intriguing still, the protein didn’t resemble any other known biological molecules except a distant relative in the lens of the eye.

Still, demonstrating that the mystery protein was the water channel took nearly a year. To devise the experiment that would make the case, Agre enlisted his colleague, physiologist William Guggino, an expert in how water moves in and out of eggs. In the 1970s, Guggino had begun exploring why fish eggs laid in salt water don’t shrivel, and frog eggs laid in pond water don’t explode. His and Agre’s guinea pig, Guggino decided, would be a frog oocyte, “one big cell, visible to the human eye, that’s very promiscuous at expressing membrane proteins on its surface.”

Sure enough, each egg doctored with Agre’s protein and placed in fresh water would swell up and burst within seconds, squirting yolk out one side. But did that alone prove the protein caused a previously impervious cell wall to suddenly sprout openings that admitted water? What if the injection itself had somehow damaged the membrane? Two refinements removed all doubt. When the researchers injected control eggs with a protein for a different kind of channel (chloride), those eggs remained intact. Even more ingeniously, they tapped the fact that mercury inhibits water movement to show that, in the presence of that element, their experimental eggs didn’t swell either. There was no longer any doubt. Agre’s protein was the water channel. He named it aquaporin.

“We put in our thumb and pulled out a plum,” he says. “It completely changed the focus of my lab. Within a week, we were getting calls from all over the world.”

It would be easy to assume that at an institution like Johns Hopkins, Nobel laureates inhabit every floor. Yet, only two full-time School of Medicine faculty members have ever received the call from Stockholm. In 1978, Daniel Nathans and Hamilton Smith shared the prize in medicine for their discovery of restriction enzymes, the so-called chemical scissors that laid the groundwork for mapping the human genome.

To a select few in the know, there were hints that Agre might be the one to break the 25-year dry spell. “Peter had been invited several times to Stockholm, including being asked to speak at the Nobel centennial in 2001,” says Dan Lane, who headed biological chemistry from 1978 to 1997, and convinced Agre in 1993 to switch to his department. “Then a member of the Nobel committee dropped in a few months ago just to chat with him. When the chemistry committee chair called to ask me if Peter was going to be here on Oct. 8, well, it certainly seemed a possibility.”

On the night of Oct. 7, Agre simply turned in as usual. “I was sleeping fine,” he says. “It’s not as though it was likely that I would win.” This time, Agre’s instinct was wrong. And throughout Hopkins ever since, the mantra has been, “It couldn’t have happened to a nicer guy.”

Notorious for downplaying his achievements, for pointing only to the serendipity of his discovery and not the years of shrewd work that preceded and followed it, Agre prefers to deflect praise with humor. He doesn’t hesitate to bring up the D he got in high school chemistry. Told that the New York Times was clamoring for an interview, he quipped, “Is my subscription overdue?” Asked about Roderick MacKinnon, the biophysicist at New York’s Rockefeller University with whom he shares the 2003 chemistry prize, Agre replied, “He’s a serious scientist. I’m hanging on.” And in response to an introduction by Chi Dang, Hopkins’ vice dean for research, Agre said, “Chi made a technical error. It was the young people in my lab who did the work. I just made the coffee.”

Don’t you believe it, says Dang. “I know Peter from way back when. I came here as an assistant professor when he was in the hematology division. I was next door when he was trying to clone this thing. Peter has this sixth sense—he captures opportunities, he’s very persistent, he does science with great depth. Young scientists today are very energetic, they want to do a hundred things. Peter wants to understand something from 3,000 miles up all the way down to the atomic level. He solved the structure of aquaporin so we really understand it. That’s the depth. That’s why he was recognized.”

Since the paper proving the existence of the water channel appeared in 1992 in Science, Agre and others have identified 11 mammalian aquaporins. The channels have turned up, not surprisingly, in the more water-permeable parts of the body: salivary, tear and sweat glands, kidney tubules, the choroid plexus of the brain where spinal fluid is produced, the ciliary epithelium of the eye where aqueous humor is formed, the moist surface tissues of the alveoli in the lungs.

What most excites physicians, though, is that aquaporin research has shifted from merely finding where else the proteins are expressed to exploring how this knowledge can be harnessed to prevent or treat disease (among the current targets: glaucoma, nephrogenic diabetes insipidus, asthma, cystic fibrosis, brain edema and congestive heart failure).

To that end, Agre has spent years studying aquaporins’ structure, and in 2000 he and others produced the first high-resolution images that show how they do what they do. “They’re shaped like an hourglass,” he says, “the first such example in nature. Nothing else is smaller than water. Aquaporin is a machine with no moving parts that lets water go through without resistance and repels everything else.”

“One of Peter’s greatest strengths is recognizing what’s important and saying, This is where we should focus,” says pulmonologist Landon King, who has worked with Agre since 1989. “It’s not blind luck. He sees things earlier than many of us. He also has this ability to take complete stories and make them understandable to almost any audi-ence. He’s uniquely qualified—and now pedigreed—to be an ambassador for science.”

And if that is a role he is to assume as a Nobel laureate, it suits Courtland Agre’s son just fine. “These things come with an obligation,” he says, “to share with young people and get them excited about science. Dad would have loved this."

Mary Ann Ayd
Hopkins Medicine, Winter 2004

http://www.hopkinsmedicine.org/press/2003/OCTOBER/031008A.HTM