An Interview with Manuel and Ann Varela: Peter Agre and Water — Water Flowing in and out of Cells – The Life and Times of Peter Agre

Mar 22, 2020 by

Peter Agre

1) Peter Agre was an American physician, a Nobel Laureate, and a molecular biologist. He studied at Johns Hopkins Bloomberg School of Public Health and Johns Hopkins School of Medicine and later became director of the Johns Hopkins Malaria Research Institute.   What do we know of his early childhood, and how did he get interested in medicine?

Dr. Peter Agre is best known for his fundamental discoveries of the water channel aquaporin and the mechanism of water transport across the living membrane. He was born on the 30th day of January, in 1949, in the town of Northfield, Minnesota, to parents Ellen Swedberg and Courtland Agre. Courtland was a chemistry professor, and Ellen was a stay-at-home mom.

In recent interviews, Agre has described his childhood as idyllic. He regarded his father as a hero who would routinely perform for him and his siblings various scientific experiments, such as changing the colors of solutions as a function of changes in pH. His father was a major influence in Agre’s decision to enter the field of medicine for a career. Agre and his brother Jim worked at a dairy farm during the summer months. Impressively, Agre became an Eagle Scout.

In high school, Agre published a sort of rouge newspaper called The Substandard, which was actually a parody of their school’s regularly sponsored newspaper, called the Roosevelt High School Standard. Facing imminent expulsion, Agre disenrolled from the school and instead finished his diploma after having attended night school.

Agre took his B.A. degree from Augsburg College, in 1970, after majoring in chemistry. He then enrolled in medical school at Johns Hopkins in Maryland. During this time, he worked as an assistant in the laboratories of Drs. Brad Sack and Pedro Cuatrecasas, where Agre had an interest in studying enterotoxin-induced diarrhea that caused dehydration and death in little children from underdeveloped countries. Dr. Agre took his M.D. degree in 1974. In 1975, Agre married Mary Macgill. They have six children, four sons and two daughters.

2) Obviously, water is a central component of the human body and is imperative for life and living. How did Peter Agre study, however, the flow of water into and out of cells, and what does this signify for our understanding of medicine?

Indeed, the requirement of water for the life of all organisms on Earth is widely appreciated. As an undergraduate university student, one of us (MFV) recalls that the very first lecture in a biochemistry course was based on the chemistry of water. We were told about the critical bond angle of 104.5 degrees that the central oxygen atom (O) makes with each of the two hydrogen (H) atoms to form water (H2O). The covalent bond lengths, 0.0965 nanometers, or 0.956 angstroms, between oxygen and its hydrogens, were critical, too. These chemical properties of the water molecule make life possible. As a consequence of these bond lengths and angles, water floats to the top as it freezes. If frozen water didn’t float, all water would eventually become frozen solid, and organisms couldn’t access the life-giving chemical to live.

Figure X. Diagram of a water molecule

Thus, water is considered a universal solvent by which all biological solutes of a chemical nature are dissolved. When water interacts with such solutes, it can form so-called hydrogen bonds, which can be weak interactions between atoms of water with those of the solutes, permitting them to be dissolved.

Before Arge’s studies of water biochemistry, it had been known physiologically that pure water could move across the biological membrane on its own. However, the water transport rates were too slow to account for the rapid water movement observed between, for instance, the kidneys and the blood. Similarly, water movement by itself across the membrane was judged too sluggish during the secretion of, for example, tears or saliva. Likewise, water must move across the membrane of red blood cells to accommodate large changes in solute concentrations in and around the blood as blood circulated in the body. For water to provide its necessary life-bestowing properties, it requires that rapid water movement must occur. This requisite for speedy transport of water across the membrane predicted that there must be a dedicated gateway, a channel, for water.

For over a century, the discovery of a water-specific channel remained elusive. It is at this point when Agre steps into the picture in 1986. He was the first scientist in history to come across the famous water channel. Agre’s water transporter became known famously as the aquaporin, and he found it quite by accident.

He had originally began a search for certain blood factors that determined the Rh factor blood type, a protein called D, which was located in the membrane of red blood cells. When they purified the protein and cloned the DNA of the gene encoding it, they instead found that it was a completely different protein, a member of the protein family called MIP, for major intrinsic protein, a channel with an unknown substrate. Upon the suggestion of a colleague, who noted that the MIPs were found in water-laden tissues and organs of the body, proposed that perhaps the MIP protein found by Agre was the elusive water channel that investigators had been searching for over many years.

The hypothesis that Agre’s MIP molecule transported water required that they directly measure the transport of water across the cell membrane. The usual process for measuring direct water transport was not available. They could readily measure, however, osmotic pressures, which were thought to be the physiological outcomes of water transport. Osmosis refers to the transport of water across the biological membrane.

Thus, the physiological test of water transport was quickly performed in frog egg cells called Xenopus oocytes. In a rather clever experiment suggested by Dr. Karl Windhager, Agre and Preston, working with Tiziana Piazza Carroll and William Guggino, injected the frog cells with mRNA encoding the CHIP-28 protein. With the novel water channel biosynthesized in the frog eggs, the CHIP-28 presumably entered into their place within the oocyte membranes. The CHIP-28-laden Xenopus eggs were placed in a solution of solutes with a very low concentration, leaving the water concentration outside the egg cells to become relatively higher, compared to water the inside, a situation known as a hypotonic solution, and the frog cells quickly exploded!

The cell explosion was caused by the novel presence of the CHIP-28 water channel. In the hypotonic solute solution, water quickly entered the cells through the CHIP-28 channel and swelled the cells to such an extent that the membrane couldn’t overcome the massive water entry. The membranes of the frog cells broke apart into pieces and couldn’t recover.

In cells with a water channel, the transport of water across the membrane moves from a high water (low solute) concentration to low water (high solute) concentration, that is, from high to low water amounts, until the water amounts are equal on both sides of the membrane. This process will stop with the water concentrations are equivalent on both sides of the membrane of a cell, a phenomenon called equilibration. Water can relocate in and out of the cell through the membrane, but the net water movement will be zero.

In the case of cell explosions, however, the hypotonic solution can make it possible that before water equilibration can occur, the membrane succumbs to the osmotic pressure exerted by the water transport, and the cell will lyse. In negative-control Xenopus egg cells, i.e., cells without the water channel, no explosions were observed in the hypotonic solution.

In hypertonic solutions, i.e., a high solute and low water concentration, water still transports across the membrane from high to low concentrations. Still, this time water moves out of the cell, thus, shrinking the red blood cell, a process called plasmolysis. Agre had demonstrated that water translocation across the membrane could be reversed through the channel.

In cells with an isotonic solute solution, the water concentrations are likewise similar, and, again, the water movement into and out of cells occurs in equilibrium with a net movement of zero.

Agre’s physiological water osmosis experiments were the first in the world to demonstrate the presence of the elusive water channel. The Nobel-prize winning work was published in the prestigious journal Science in April of 1992. Agre’s water transport mechanism is featured on the first page of a current popular biochemistry textbook. He was featured in Albert Lehninger’s famous book as having applied biochemical approaches to discover the then elusive water channel in the biological membrane.

The aquaporin protein has medical importance. For example, water transport occurs in the kidneys to collect water and maintain a proper water balance in the human body. Aquaporins function in the brain to export any excess of water during brain swelling, preventing the death of the individual with brain edema. Another example lies with the glands for sweat secretion, which is important for helping to uphold the correct body temperature. Any deviations from 37°C would almost certainly be detrimental to the functioning of the body and could lead to an ability to maintain the life of an organism.

3) Aquaporin—what is it, and how did it impact Agre’s research?

The aquaporin protein is the water channel that Agre serendipitously discovered when he had attempted to isolate the Rh blood factor. Instead, he discovered the CHIP-28, which was later renamed as aquaporin (AQP-1), to describe its functional properties better. The protein was integral to (i.e., embedded in) the membrane, and it formed a pore, which allowed water to move through it such that the water could translocate across the membrane. In the figure shown, the membrane-bound aquaporin is cut in half, revealing the water pore or channel itself. The cell membrane is depicted by two layers of phospholipids. The water molecules are shown in the middle of the channel pore structure, and you can see how the water has specific access to either side of the membrane. The centrally-located and narrowest size of the pore is about 2.8 angstroms, which severely limits the sizes of molecules that can pass through the pathway. Further, the pluses indicated in the channel are plus-charged amino acids, like arginine, which effectively repel positively-charged hydrogen atoms (protons) or other plus-charged ions, such as sodium or lithium. These properties permit the aquaporin to be highly selective for the water, leaving out virtually all other potential substrates. The mechanism is also known as a selectivity filter.

Figure x The water channel.

In molecular terms, the AQP-1 aquaporin can be shown as a so-called ribbon structure. In the figure below, aquaporin is depicted without its associated membrane for clarity and in which the α-helices are shown as dark gray spirals. The AQP-1 water channel harbors six such α-helix spiral segments. These segments are also known as trans-membrane α-helices. Each of these transmembrane helices contains an amino acid sequence triplet of asparagine-proline-alanine (Asn-Pro-Ala) that are highly conserved evolutionarily speaking. Between the six spirals are thin-looking loops that interconnect the α-helices. These thin loops are also referred to as β-structures.

Figure x Aquaporin Molecular Structure.

Several of the distinctive aquaporins, however, such as AQP-7, have been shown to have other substrates, such as glycerol or urea. Whether transporting water or other small molecules, the aquaporins can be regulated by their loop structures mentioned above. These loops are thought to serve as gates, which also permit strict permeation of water. The gate mechanism is phosphorylated, which aids in closing the channel to prevent leakage.

As the water permeates through the channel portion of the pore, they move through the channel in a single file process, at about a million waters per second on average, with about a billion water molecules per second in the case of AQP-1. A given red blood cell harbors about 200,000 AQP-1 individual molecules in its membrane.

After Agre’s discovery of the AQP-1 in humans, an additional nine distinctive water channels were discovered, each given a new number, e.g., AQP-2, AQP-3, AQP-4, etc. In humans and other mammals, the aquaporins are found in red blood cells, kidneys, blood capillaries, eyes, salivary glands, brain, liver, testis, stomach, and lungs. The aquaporins were discovered in bacteria, plants, fungi, insects, amphibians, and non-human animals. All groups living organisms on Earth have aquaporins. Further, the aquaporins were highly similar in sequence and structure, attesting to their homology, i.e., that they shared a common ancestral origin. Thus, the widely conserved nature of water transporters is not surprising, considering that all life on the planet needs water.

4) Two different aquaporins were later discovered, and it was found they had much to do with kidney function and the urine and water. How does this all work in the kidneys?

Indeed, the kidney functions to filter out toxic substances from the blood, to remove waste products, and to maintain proper ion balances. Kidneys also regulate the volume of the body’s fluids, solute concentrations (osmolarity), ion amounts, proton concentrations, and make needed hormones. Kidney damage or failure can lead to serious consequences, like an unhealthy build-up of toxic compounds, waste materials, or kidney stones.

Of the 11 known aquaporins found in mammals, seven are found in the human kidneys: AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP8. Two of these, AQP1 and AQP2, are of prime importance.

The AQP1 protein was the first to be studied extensively. It can be found in many anatomical areas of the kidney, such as the proximal convoluted tubules, descending thin limbs, and in the basolateral membranes of the kidney’s brush borders. Together, the AQP1 water channels in these areas serve to conduct massive water resorption in the kidney. It is a life-maintaining process.

In the early 1990s, the AQP2 protein was discovered to reside in the apical plasma membrane of the kidney. Prior to this discovery, it had been known that the antidiuretic hormone vasopressin controlled the delivery of vesicles inside cells of the renal collection duct to the apical surface membrane of these so-called principal kidney cells. The hypothesis was that vasopressin mediated this membrane shuttle system of vesicles to deliver AQP2 back and forth so that the channels could provide quick water transport as needed. To the present day, this water transport system is an active area of biochemical and biomedical research.

5) At one point, he shared the Nobel Prize with another American. What exactly did the two do to be recognized for the Nobel Prize?

Peter Agre took the Nobel for having discovered the water channel, which he demonstrated mediated the translocation of water molecules across the cellular membrane. Dr. Agre shared the Nobel Prize for chemistry in 2003 with Dr. Roderick MacKinnon, who was then at the Rockefeller University, in Rochester, New York.

MacKinnon first discovered the structure of the membrane-laden channel for the potassium ion (K+) in the bacterium called Streptomyces lividans. Then, he used his new protein structure to evaluate its mechanism of channel function in the microbe. In so doing, he discovered the so-called ion selectivity filter inside the passageway of the K+ channel.

In the mid-1990s, MacKinnon and collaborators observed that the K+ channel favored potassium ion translocation over that of the sodium ion (Na+) about 10,000 times better. They set out to elucidate how the so-called ion selection, K+ preferred over Na+, came about in the K+ channel itself. They observed that when cells harboring the K+ channel were exposed to both Na+ and K+ ions at the same time, the two ions entered the selectivity filter inside the channel. However, only the K+ could move past the filter because it was just narrow enough to accommodate its passage through the gateway and move across the membrane to exit to the other side of the cell. The Na+ ion, on the other hand, is just slightly larger than K+ ion (just 0.4 angstroms larger!), and the Na+ ion cannot, therefore, pass through the filter. The Na+ is simply too large.

MacKinnon further enlightened the ion channel function. He and colleagues discovered that certain amino acids within the ion selectivity filter of the channel provided binding specificity. In the ion selectivity filter itself was lined with specific amino acids that provided a K+ binding site, consisting of oxygen atoms. The amino acids tyrosine and glycine provided the required oxygens.

In the mechanism discovered by MacKinnon, the ion-binding to its dedicated filter necessitated that the K+ ion first loses its surrounding water layer, called a hydration shell. The oxygen atoms in the ion selectivity filter facilitate the water loss from K+ ion. When the hydration shell is shed, all eight of its water molecules come off of the K+ ion, and the ion then specifically makes chemical contacts with the ion selectivity filter, permitting the ion to move through the gateway of the channel. This work, the K+ channel isolation, and its ion selection mechanism earned MacKinnon the Nobel.

In the Nobel work of Agre, he discovered the aquaporin water channels. He had initially set out, however, to study the so-called Rh blood group antigens, also known as protein D or Rh D, for Rhesus monkey protein D. These D factors are denoted by a plus or minus sign. The signs are used to indicate whether an individual possesses (Rh+) or lacks (Rh) the antigen on their red blood cell membrane surfaces. First, Agre and his colleagues, Andrew Asimos, Ali Saboori, and Barbara Smith, obtained human blood and extracted as many of the membrane-bound proteins as they could. Next, they labeled the proteins with radioactive iodine, called 125I, and, using an antibody directed against the Rh D antigen, succeeded in purifying a candidate protein in 1987.

Unfortunately, the investigators found out that their protein was present in meager amounts. Then, Agre and Smith, together with Bradley Denker and Francis Kuhajda, attempted in 1988 to characterize the protein mixture using electrophoresis gels to separate the individual proteins based on their charges and sizes. To their surprise, an electrophoretic band on the gel at a molecular size of 28,000 Daltons (Da) failed to stain with Coomassie blue, a reagent known to bind proteins! This low protein turnout and the failure to stain with the reagent led the biochemists to believe that the purified radioactive protein was merely a fragment of their total desirable Rh D protein. They speculated inaccurately that the pure protein played a structural role in the shape of the red blood cell, calling it a membrane skeleton.

Next, in 1991, Smith and Agre used a better antibody to detect the 28-kDa protein candidate and found that it had a sugar component to it that made it more substantial, on the order of 40- to 60-kDa. The sugar-protein combination was referred to as a glycoprotein or an N-glycosylated protein. They could knock off the sugar part if so desired, bringing back the smaller 28-kDa part. The amino acids of the protein part were hydrophobic in their nature, indicating that they could associate with the corresponding hydrophobic membrane of the red blood cells to which the protein belonged.

Then, the Agre laboratory realized they were working with repeat units of the protein, forming complexes with each other to form an oligomer. In Agre’s case, the four sub-units came together to build a larger version, called a tetramer. During this time, in 1991, Gregory Preston and Agre cloned the gene that encoded the 28-kDa monomer, and they determined the DNA sequence of their candidate gene. They entered their gene sequence into the more extensive DNA sequence databases available at the time. Next, Preston and Agre asked the computer to deduce the amino acid sequence encoded by their gene, and what they found surprised them completely!

Agre’s protein sequence showed similarity with poorly characterized channel proteins, called major intrinsic proteins (MIP) found in eye lenses from cows! Unfortunately, the Agre laboratory was still at a loss regarding what an integral membrane protein in the human red blood cell had to do with a cow eye lens and, importantly, what the function was of their 28-kDa protein! That is, Agre and colleagues did not know what the substrate of their new channel was—we now know it was water! But at the time, in the early 1990s, it was not so clear cut. It was an orphan channel, that is a transporter without a known substrate. Thus, they called their red blood cell protein, CHIP-28, for channel-like integral protein.

Then, the CHIP-28 protein, found in blood, was discovered within the kidney, an organ that also required fast water transport across the membrane. It was this finding that Dr. John C. Parker, a colleague of Agre, to predict that the CHIP-28 was the elusive water channel! The Agre laboratory then conducted the necessary water transport experiments described above and demonstrated that their CHIP-28 was indeed the long sought after water channel, later called aquaporin.

6) Apparently, he was also involved with the American Association for the Advancement of Science- what position did he hold, and what were his contributions to that organization?

Peter Agre was president of the AAAS, and starting in 2009, he served two years in this capacity. Established in the U.S. in 1848, the AAAS became an international and scientifically-based non-profit organization for scientists. It is devoted to the advancement of communication between scientists and the general public. One main goal was to promote scientific integrity and the responsible use of science technology. It is also meant to foster scientific education and research. The AAAS oversees several prestigious journals, such as Science, one of the world’s premier scientific journals.

During his tenure as president, Agre was active in the Center for Scientific Diplomacy within the AAAS organization. In this vein, he formed a scientific delegation and made a series of diplomatic visits to foreign countries that held somewhat adversarial relations with western countries, like the U.S. Argre’s main goals in this effort was to foster better scientific relationships with the scientists housed in Burma (Myanmar), Cuba, North Korea, and Iran. In each of these countries, Agre was reported to have made tremendous strides towards fostering better scientific collaboration and in reducing diplomatic tensions.

7) What have I neglected to ask about this famous scientist who investigated the transportation of water into and out of the cells and the cell membrane?

Dr. Agre interned at Case Western Reserve and conducted a post-graduate fellowship in the fields of oncology and hematology at North Caroline Memorial hospital. Then, he moved to work in the laboratory of professor Vann Bennett at Johns Hopkins. In 1992, Agre became full professor Johns Hopkins, and in 2003, he became director of the Malaria Research Insitute.

Agre became a full-fledged member of the National Academy of Sciences in 2000, a prestigious honor. He joined the American Academy of Arts and Sciences in 2003 and the American Society for Microbiolgy in 2011.

Agre once made an appearance on The Colbert Report, where he discussed his part in establishing the Scientists and Engineers for America (SEA). During the television broadcast, Agre noted the serious decline of scientific knowledge in Americans.

For further information regarding this famous biochemist, go to:

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