The Floodgates Open for Aquaporins

WATER IN TRANSIT Courtesy of Bing Jap Close-up view of the AQP1 water channel derived from crystal structure. Residues defining this region are depicted in ball and stick (Grey, carbon; blue, nitrogen; red, oxygen; yellow, sulfur). The water molecule located just below the constriction region is shown. Histidine protrudes into the pore and is a key residue for establishing water selectivity. For some, the road to discovery is a four-lane highway lined with sign-posts warning what lies ahead. For Peter Agre, the Johns Hopkins University researcher who accepted the 2003 Nobel Prize for chemistry last month, it was more like "driving out on a gravel road in a remote part of western Maryland and suddenly coming to a large city" – hard to miss and yet completely unexpected. Since Agre and his group cloned aquaporin 1 (AQP1) in 1992, hundreds of these previously unknown channels have been discovered in plants, animals, and lower organisms; researchers have made progress toward understanding their structure and mechanism, and how faulty regulation can lead to disease. Yet, as with many other proteins, a trickle of information soon turns into a downpour as it becomes apparent that functions may be much more diverse than originally anticipated. Before their discovery, the existence of water-specific channels was "pretty controversial," says Agre. Most physiologists asserted that the membrane itself had enough intrinsic permeability to account for water movement between tissues. Soon after their discovery, aquaporins were linked to multiple clinical syndromes involving defective water homeostasis. In the kidney, for example, faulty AQP2 regulation leads to nephrogenic diabetes insipidus (NDI), in which the kidneys cannot concentrate urine and patients easily become dehydrated. In contrast, AQP2 overabundance may lead to fluid retention in congestive heart failure and pregnancy. In the brain, AQP4 is involved in monitoring fluid levels as necessitated by the skull's constraints. Aquaporins also regulate water content in the eye, and defects can lead to congenital cataracts.1 Yet researchers increasingly report observations that aquaporins do more than regulate water homeostasis. Harvard University's Dennis Brown studies vasopressin receptor-mediated regulation of AQP2 in the kidney. His group has revealed how receptor activation increases cell-surface levels of AQP2, enhancing water permeability as needed. These studies may lead to a treatment for NDI, though Brown notes that the kidney contains two other aquaporins whose roles are still unknown. Aquaporins also are found in such diverse cell types as brain astrocytes and gastric parietal cells, where potential functions are not obvious. "Nobody knows exactly what it is doing in all these tissues," Brown says. "My prediction is that we'll be finding other functions [perhaps as] receptors or as cell-cell adhesion molecules." Another aquaporin, AQP4, has mysteriously turned up in muscle cells. Its absence is a common feature of several muscular degenerative diseases, including Duchenne muscular dystrophy, but it is unclear whether this absence contributes to the disease, says Stanley Froehner, University of Washington. Froehner's group studies dystrophins, a family of muscle proteins. His team became interested in aquaporins when it was found that α-syntrophin, an accessory protein to dystrophin, serves as the membrane anchor for AQP4 in certain astrocytes lying at the blood-brain barrier. Together with Ole Petter Ottersen, University of Oslo, they have shown that α-syntrophin knockout mice are resistant to cerebral edema, a life-threatening flow of water into the brain, because they lack proper placement of AQP4 in these cells.2 While this work marks AQP4 as a potential drug target, its role in normal brain function remains unclear. WET SERENDIPITY From the start, aquaporins have defied expectations. Agre and his group originally set out to purify an unrelated protein from red blood cells, but their attention was caught by a mysterious, highly abundant protein contaminant, which turned out to be AQP1. Because researchers used the red blood cell extensively as a model experimental system, the discovery of a major new protein in its membrane was highly unexpected, he says. After purifying it and cloning the cDNA, their first clue that it might be a channel was its amino acid sequence, which predicted six membrane-spanning α-helices. Moreover, sequence homology showed that it was related to MIP26, a major lens protein involved in fluid uptake, as well as to a protein in plants whose expression is induced by water deprivation. Based on these clues, Agre and his group set out to determine whether they had isolated a water channel, using a somewhat unusual experimental system: the exploding Xenopus oocyte. Normal oocytes survive quite well in a low-salt environment. But the presence of a water channel in the oocyte membrane spells trouble, because water can then rush down the osmotic gradient into the high-salt oocyte interior. Eventually, the oocyte swells and bursts due to water influx. Agre's group injected oocytes with mRNA transcribed from their cDNA clone, and numerous videos of badly damaged oocytes confirmed their suspicions that they had identified a water channel protein.3 STRUCTURE OF A SIEVE These newly discovered channels displayed some intriguing properties. For example, while highly permeable to water, aquaporins prevent other small molecules, such as urea, ions, or even protons, from passing through. The desire to understand how such rapid permeation could be coupled with such high specificity led to a "friendly rivalry" among structural biologists to solve structure, says Bing Jap, Lawrence Berkeley National Laboratory. Two high-resolution versions resulted. Robert Stroud's group at the University of California, San Francisco, crystallized the glycerol facilitator, GlpF, which transports both glycerol and water, from Escherichia coli.4 A year later, Jap's team produced a high-resolution structure for AQP1.5 Most recently, Stroud's group has published the structure of AQPZ, also from E. coli.6 These structures show that aquaporins consist of a right-handed bundle of six highly tilted, membrane-spanning α-helices. The pore's hour-glass shape has wide extracellular and intracellular vestibules, joined by a long narrow region that acts as the selectivity filter. In native tissues, four aquaporin monomers team up to form a tetramer, but each monomer contains a separate water pathway. The high-resolution AQP1 structure also contained a bonus: four almost perfectly resolved water molecules caught in the act of traversing the pore. According to Jap, water is not usually visible in protein crystal structures unless it is very tightly bound. These waters were thus bound in a highly specific fashion that reflected the channel's mechanism. "From the structure itself, you already know exactly how the channel works," he says. The pore's high selectivity arises from two features. First, it is very narrow, excluding all but the tiniest molecules, and ensuring that ions cannot enter unless they shed their bulky hydration shells. Second, it is highly hydrophobic, with the exception of a few strategically placed hydrophilic sites where water molecules can form hydrogen bonds. The relatively long distances between these hydrophilic sites prevent ions, including protons, from traversing the pore's entire length. This high hydrophobicity also provides rapid permeation, since it discourages water molecules from lingering inside for very long, says Jap. SUBSTRATES, SUBSTRATES EVERYWHERE Perhaps the most controversial proposed new role for aquaporin is Yale University researcher Walter Boron's* finding that AQP1 can conduct CO2.7 In a scenario reminiscent of aquaporin's original discovery, other investigators have questioned the need for a specific CO2 channel, since most membranes are highly gas permeable. Boron admits that under normal conditions, CO2 membrane permeation is not "a limiting factor" in red blood cells where AQP1 resides. However, he points out that CO2 permeation could become a limiting factor under stress conditions, such as during peak athletic performance or for those living at high altitudes. These ideas have been given credence by the recent finding that the tobacco aquaporin, NtAQP1, also conducts CO2.8 "Photosynthesis is CO2-limited," he points out, supporting a physiological relevance for this function. Bert de Groot, who studies aquaporin mechanisms together with colleague Helmut Grubmüller at the Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, writes in an E-mail: "Based on its size and physiochemical properties, it is well conceivable that CO2 might permeate aquaporin [-like] pores." He further suggests that the great need for CO2 in plants might then explain why "plant genes encode for hundreds of aquaporins, whereas mammals only have something on the order of 10." Ventures into this bustling city of membrane proteins already have revealed surprises. Further exploration may show that these multipurpose channels are even more pervasive. As Harvard's Brown remarks, "Perhaps the name aquaporin was a little hasty." Only time will tell. Megan Stephan dr.meganstephan@att.net is a freelance writer in Cheshire, Conn. *The author performs research in a Yale University laboratory that is not affiliated with Dr. Boron's work.

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The Floodgates Open for Aquaporins

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How Do Embryos Know How Fast to Develop

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