Aquaporins: Channels for Advanced article Article Contents the Molecule of Life • Introduction • A Discovery Well Worth a Nobel Prize Uwe G Hacke, University of Alberta, Edmonton, Canada • Structure, Permeation and Substrate Specificity Joan Laur, University of Alberta, Edmonton, Canada • Regulation • Roles of Aquaporins in Different Life Forms • Future Directions • Acknowledgements Online posting date: 15th February 2016 Liquid water has unique properties that make it The aquaporin protein family is ancient, and aquaporins can be a universal solvent. Water is an essential compo- found in a wide range of organisms, from unicellular bacteria nent of almost all physiological and biochemical to giant Sequoia trees. All aquaporins are recognised by shared, reactions; therefore, its presence is required every- conserved structures. The structure of the channels is impor- tant because it determines which molecules can pass, which are where within an organism. The circulatory systems excluded and at which rate molecules can move through the pores. of animals and the vascular system of plants move fluids over long distances. In the tallest trees, water moves across a height gradient of 100 m or more, A Discovery Well Worth a Nobel thereby connecting roots and distant leaves. On a cellular and tissue level, water movement is facili- Prize tated by intrinsic membrane proteins called aqua- Transmembrane water flow was long believed to depend entirely porins. These water channels are found in all life on the permeability of the lipid bilayer and its composition. But forms. Aquaporins have been mostly studied in the high water permeability of certain membranes could not be mammals and plants, where water channels play fully explained until Peter Agre’s group (Smith and Agre, 1991; important physiological roles. This article gives an Agre et al., 1993) isolated and characterised the first human aqua- overview of the discovery, structure and regulation porin, CHIP28, later renamed AQP1. For decades, people have of aquaporins. Their roles in different life forms are speculated on the existence of microscopic pores that facilitate discussed. movement of molecules like water across biological membranes (Stein and Danielli, 1956). Rapid water flux was observed in toad bladders, mammalian kidneys and red blood cells. It was also found that high rates of water movement could be blocked in Introduction a reversible way by cytotoxic mercury reagents, suggesting the protein nature of what was later called aquaporins (Macey and While the lipid bilayer allows the slow diffusion of water Farmer, 1970; Macey, 1984). Finally, Wade et al. (1981) formu- molecules (see also: Lipid Bilayers), only the presence of lated the membrane shuttle hypothesis based on observations of a dedicated water channels can explain substantial water flow concomitant increase in cell permeability and the massive reloca- rates across some cell or subcellular membranes. Aquaporins are tion of protein aggregates from intracellular vesicles to the plasma integral membrane transport proteins, and they facilitate water membrane. What was the molecular identity of those proteins? movement in cells, tissues and entire organisms. The proper dis- It was also in the 1980s that Agre started working on the Rh tribution of the most abundant molecule in cells and living tissues blood group antigens. In the early years, his team had isolated is fundamental to life. In addition to water, certain aquaporins two membrane proteins of 32 and 28 kDa from red blood cells. also facilitate the transport of small solutes such as glycerol and The 28 kDa protein was found in spectacular quantity: 200 000 perhaps even gases like carbon dioxide. Molecules move through copies per red blood cell (Agre, 2004). The partial analysis of its the channels in response to osmotic/concentration gradients. sequence showed its close relation to proteins found in the kid- ney, eye lens, brain cells and also bacteria and plants; and several features suggested it was a membrane channel. The protein, tem- eLS subject area: Plant Science porarily named CHIP28 (for channel-like integral protein of 28 How to cite: kDa), was the subject of many discussions; but finally it was sug- Hacke, Uwe G and Laur, Joan (February 2016) Aquaporins: gested that it might be a water channel (Carbrey and Agre, 2009). Channels for the Molecule of Life. In: eLS. John Wiley & Sons, This hypothesis was soon tested by expressing CHIP28 in frog Ltd: Chichester. oocytes. This was a useful experiment, because oocytes nor- DOI: 10.1002/9780470015902.a0001289.pub2 mally show low water permeability. Oocytes expressing CHIP28 eLS © 2016, John Wiley & Sons, Ltd. www.els.net 1 Aquaporins: Channels for the Molecule of Life Control CHIP28 sequence cRNA Test Xenopus Oocytes Figure 1 Expression of CHIP28 water channel activity in Xenopus oocytes. Oocytes obtained from female Xenopus are injected with in vitro transcribed RNA of CHIP28. After a period of incubation, the water permeability of test oocytes expressing the protein and of control oocytes is tested in distilledwater. Almost immediately, ‘the test oocytes were highly permeable to water and exploded like popcorn’ (Agre, 2004). immediately swelled and exploded when transferred in distilled the answer is ‘no’; exceptionally high flow rates are possible water (Figure 1). Permeability to water was drastically increased. because of fine-tuned interactions between water molecules and The first water channel protein had just been functionally charac- the molecules forming the channel (Kozono et al., 2002; Eriksson terised ‘following the well-known scientific approach known as et al., 2013). sheer blind luck’ (Agre, 2004) and years of hard work. Aside from the central NPA constriction, aquaporins contain an outer constriction. This aromatic/arginine (ar/R) constriction region creates the narrowest section of the channel and constitutes Structure, Permeation a major checkpoint for solute permeability (Figure 2,dashed and Substrate Specificity ellipse). By mutating the ar/R filter, Beitz et al.(2006)were able to modify the water selectivity of AQP1 to allow urea, Aquaporins belong to the highly conserved major intrinsic protein ammonia and glycerol permeation. In aquaglyceroporins, the (MIP) super family (Danielson and Johanson, 2010). MIPs exist ar/R constriction region is wider than in orthodox water pores, as tetramers. Each subunit is behaving as a single aqueous pore. allowing the passage of larger molecules such as glycerol. Subunits share the same hourglass structure consisting of six transmembrane domains connected by several loops (Figure 2). Two of these loops fold back into the membrane and overlap. Regulation The overlapping loops each contain the conserved signature motif asparagine, proline, alanine (NPA). Located in the narrow centre Some aquaporins contain an additional energy barrier to water of the pore, the NPA region represents a key feature for water permeation, which allows these channels to be opened or permeation. The centre of water-selective pores has a minimum closed. In the spinach aquaporin SoPIP2;1, the phosphorylated diameter of 2.8 Å, almost matching the diameter of a water status of highly conserved amino acids, Ser115 and Ser274, molecule (Kozono et al., 2002). This means that water molecules controls the open/close conformation (gating) of the channel move through the centre of each pore in single-file configuration. (Törnroth-Horsefield et al., 2006). Such modifications, and also The water molecule passing the NPA motifs undergoes a transient the changes in pH, osmolarity and membrane tension, may lead reorientation as it forms hydrogen bonds between its oxygen and to the physical obstruction of the channel (Hedfalk et al., 2006). the partial positive charges of pore-lining asparagine residues. Other than gating, two other major modes of aquaporin regu- As a result of this and other features of the channel, protons are lation have been actively investigated: modification of their sub- unable to permeate the pores. The conserved NPA motifs’ partial cellular localisation and changes in gene expression. The shut- charges also play a key role in enhancing flow rates through the tle hypothesis (Wade et al., 1981), for instance, introduced the channel (Farimani et al., 2014). idea of water channel trafficking as a regulation strategy. The Despite the fact that water channels are only slightly wider than vasopressin-induced relocation of AQP2 that was observed in the water molecules that pass through them, they are enormously kidney cells is still intensively studied (Nedvetsky et al., 2009), efficient. Computer simulations suggest that a single water chan- and several other examples of protein transfer to the membrane nel allows the passive passage of more than one billion water have been reported (Ishikawa et al., 1999; Vera-Estrella et al., molecules per second; this is considerably faster than transport 2004; Boursiac et al., 2008;Luuet al., 2012). in some ion channels. With such extraordinary permeation rates, Another strategy to alter the number of proteins present in aquaporins are ideal channels to distribute water within organs or the membrane is to modify the level of gene expression, an tissues. The channels have to be narrow to be selective, but should approach used in the field of plant biotechnology in an effort this not come at the cost of slowing down transport? Apparently, to improve traits like drought resistance (Martre et al., 2002; 2 eLS © 2016, John Wiley & Sons, Ltd. www.els.net Aquaporins: Channels for the Molecule of Life Extracellular side HE H2H H4 H3 ar/R filter NPA signature motifs H6 H5 H1 HB N C Intracellular side Figure 2 All aquaporin proteins share a common hourglass structure. They are composed of six transmembrane helices (H1–H6) connected by five loops (LA–LE); both N-andC-termini are located on the cytoplasmic side of the membrane. Two short helical domains (HB and HE) of LB and LE form a seventh ‘broken’ helix; they both contain a signature motif NPA (asparagine, proline, alanine) located in the middle of the pore. The aromatic/arginine constriction site (ar/R) is located closer to the extracellular side.