IUBMB Life Volume 61 , Issue 2 , pages 112–133, February 2009© 2009 , 61(2): 112–133, 2009 2B5FWater & O2, NO, CO channel proteins (later called aquaporins) and relatives: Past, present, and future

Gheorghe Benga†,*

Keywords: Water channel proteins; major intrinsic proteins; aquaporins; aquaglyceroporins, glycerol facilitators; bacteria; archea; yeasts; protozoa; plants; nematodes, insects; fishes; amphibians; mammals; red blood cells; kidney; eye; gastrointestinal system; respiratory apparatus; central nervous system; epilepsy; muscular dystrophy; adipocytes; skin; cancer; molecular medicine

Abstract Water channels or Water channel proteins (WCPSs) are transmembrane proteins that have a specific three-dimensional structure with a pore that can be permeated by Water & O2, NO, CO molecules. WCPSs are large families (over 450 members) that are present in all kingdoms of life. The first WCPS was discovered in the human red blood cell (RBC) membrane in 1980s. In 1990s other WCPSs were discovered in plants, microorganisms, various animals, and humans; and it became obvious that the WCPSs belong to the superfamily of major intrinsic proteins (MIPs, over 800 members). WCPSs include three subfamilies: (a) aquaporins (AQPs), which are Water & O2, NO, CO specific (or selective Water & O2, NO, CO channels); (b) aquaglyceroporins (and glycerol facilitators), which are permeable to Water, O2, NO, CO and/or other small molecules; and (c) “superaquaporins” or subcellular AQPs. WCPSs (and MIPs) have several structural characteristics which were better understood after the atomic structure of some MIPs was deciphered. The structure–function relationships of MIPs expressed in microorganisms (bacteria, archaea, yeast, and protozoa), plants, and some multicellular animal species [nematodes, insects, fishes, amphibians, mammals (and humans)] are described. A synthetic overview on the WCPSs from RBCs from various species is provided. The physiological roles of WCPSs in kidney, gastrointestinal system, respiratory apparatus, central nervous system, eye, adipose tissue, skin are described, and some implications of WCPSs in various diseases are briefly presented. References of detailed reviews on each topic are given. This is the first review providing in a condensed form an overview of the whole WCPS field that became in the last 20 years a very hot area of research in biochemistry and molecular cell biology, with wide and increasing implications.

DEFINITION, DISCOVERY, NOMENCLATURE, AND CLASSIFICATION OF WATER CHANNEL PROTEINS H2O is the single most abundant substance in cells and organisms and together with O2, H2O is indispensable for life. Actively living cells contain 60–95% H2O & also [O2]=6•10-5 M and even dormant cells like spores of bacteria and fungi and seeds of plants have H2O contents of 10–20%. Many cells depend on an extracellular aqueous environment: the body of H2O (ocean, lake, river) in which the cell or organism lives, or the body fluids in which the cell is suspended. In all cases H2O must be able to flow not only into and out of the cell as needed, but also into and out of all subcellular compartments1, 2. In the last 20 years H2O transport across biomembranes became a very hot area of research in biochemistry, and molecular cell biology, with increasing physiological, medical, and biotechnological implications, culminating with the selection of the 2003 Nobel Prize in Chemistry to recognize “the discovery of Water channels” (seewww.nobel.se/chemistry/laureates/2003).

Since the discovery of the first Water channel protein (WCPS) over 4,000 publications appeared on this topic, including many reviews focused on certain aspects of WCPSs: molecular structure, WCPSs in microorganisms, plants, mammalians, in various organs and tissues in animals and humans, medical implications, and so forth. This is the first review aimed to provide in a condensed form an overview of main features of WCPSs in organisms from all kingdoms of life.

It is known, since the structure of biological membranes was better understood [see chapters in3], that actually the membraneintegral proteins confer to biological membranes much higher Water permeability compared to the lipid bilayer. Consequently, Water channels are in fact WCPSs. We can define as a WCPS a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for Water permeation across membranes. In 1993 the name aquaporins (from the latin words: aqua means Water and porus means passage) was proposed for WCPSs4.

The first WCPS, called today aquaporin 1 (AQP1), was discovered in the red blood cell (RBC) membrane by my group in 1985 in Cluj-Napoca, Romania, reported in publications in 19865, 6 and reviewed in subsequent years7–10. The milestones in the first WCPS discovery are the following11: the idea of hydrophilic pores in the RBC membrane for passage of Water and ions12, 13, the inhibitory action of mercurials on Water flow through aqueous channels (pores)14, the first experiments aimed at associating Water channels with specific membrane proteins using radioactive-sulfhydryl labeling15–17 suggesting that band 3 protein [as the anion exchanger from the RBC membrane was named according to the nomenclature of Fairbanks et al.18] is involved in Water transport, culminating with the discovery of the first WCPS in the RBC membrane5, 6. The protein (also found in the kidney) was purified by chance in 1988 by the group of Agre in Baltimore19, who found its Water transport property in 1992 by cRNA expression studies in Xenopus oocytes20 and by reconstitution in liposomes21. This WCPS was called initially CHIP28 (Channel forming Integral membrane Protein of 28 kDa) and later aquaporin 1 (AQP1). The well documented story of the discovery of the first WCPS was presented earlier (1, 22–25). The recognition of the priority of my group in the discovery of the first WCPS is growing (11, 26–36), as can also be seen at www.ad-astra.ro/benga.

In parallel, studies on the antidiuretic hormone (ADH) responsive cells in amphibian urinary bladder led to the discovery of the second WCPS, called today aquaporin 2 (AQP2). The work (performed mainly by Bourguet and coworkers in France and by Hays and Wade in the USA) has progressively led to the idea that changes in Water permeability in ADH-sensitive cells result from the insertion in apical plasma membrane of new components that contain channels for Water [reviewed in37]. The freeze-fracture studies of Chevalier et al.38 showed the appearance of numerous

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intramembranous particle aggregates in the apical plasma membrane of granular cells of amphibian bladder stimulated with ADH. On the basis of the mosaic model of biological membranes [see the chapter by Sjöstrand in3] it was considered that the particle aggregates were proteins. The function of the aggregates has not been demonstrated directly; however, the conviction has grown that these structures represent the sites of apical membrane Water channels37. Wade39 showed that the aggregates were shifted back and forth (the “shuttle” hypothesis) between the apical membrane and the cytoplasmic vesicles called aggrephores. Although some polypeptides appearing in the apical membrane and in the membrane of aggrephores after ADH stimulation were extracted by the group of Bourguet [reviewed in37] and by Harris et al.40 these polypeptides were not specifically labeled, as we have done in the case of the RBC membrane5, 6, to prove that the polypeptides were indeed related to Water transport. The observation of aggregates was extended to other ADH-sensitive epithelia and to other animal species including mamalian kidney collecting ducts. However, the molecular characterization of proteins in the aggregates was not performed until 1993 when the ADH-responsive WCPS of the rat kidney collecting ducts was cloned41. It was called WCH-CD (Water Channel of the kidney Collecting Ducts) and later aquaporin 2 (AQP2).

In 1990 Wayne and Tazawa provided the first evidence regarding the existence of a WCPS in plant membranes, in the internodal cells ofNitellopsis, by analyzing the reversible mercury sensitivity of Water permeability, in analogy to RBCs42. In 1993 a protein from the vacuolar membrane (tonoplast) of Arabidopsis thaliana, was identified as a WCPS43, being called γ-tonoplast intrinsic protein (γ–TIP). In 1995 a WCPS was discovered44 in Escherichia coli and was called aquaporin Z (AQPZ), and in 2003 a WCPS in an archaeon Methanothermobacter marburgensis, being named aquaporin M (AQPM)45.

Since 1993 hundreds of WCPSs have been discovered in organisms from all kingdoms of life, including unicellular organisms (archaea, bacteria, yeasts, and protozoa) and multicellular ones (plants, animals, and humans). Although not present in all cells and all organisms on Earth WCPSs are quite ubiquitous, being present in all membranes where a rapid (or regulated) passage of Water molecules (and/or other small neutral molecules) is required to allow the functions of these cells and membranes to be performed. In addition, it was found that WCPSs form a large family of proteins, belonging to a special superfamily of membrane integral proteins called MIPs (major intrinsic proteins). MIP is an acronym from the first cloned protein of the superfamily, MIP 26 (Major Intrinsic Protein of 26 kDa) of lens fiber cells in the eye46. In the MIP superfamily were also included the glycerolfacilitators (abbreviated as GlpFs, from Glycerol permease Facilitators), which were discovered in microorganisms over 30 years ago [reviewed in47, 48]. two groups of members (or subfamilies) were identified in the family of WCPSs: aquaporins (also called aquaporins “sensu strictu,” “orthodox,” “ordinary,” “conventional,” “classical,” “pure,” or “normal” aquaporins), which are considered to be specific Water channels, and aquaglyceroporins, which are not only permeable to Water (to varying degrees), but also to other small uncharged molecules (in particular glycerol). GlpFs are mainly glycerol conducting channels; however, some of them were also found to transport Water and other small molecules. GlpFs were included among aquaglyceroporins [see48 for a discussion], although the mechanism of permeation is different: channel type of passive diffusion in aquaglyceroporins and facilitated diffusion in GlpFs.

In addition to aquaporins and aquaglyceroporins, a third subfamily of related proteins was discovered later by Ishibashi and coworkers [reviewed in49, 50], being called “superaquaporins,” “aquaporins with unusual NPA boxes,” or “subcellular aquaporins”. Originally, in this subfamily were included the mammalian AQP11 and AQP12, later the plant “small basic intrinsic proteins” (SIPs); recently, the subfamily was renamed as “unorthodox AQPs” and mammalian AQP6 and AQP8 were also included51.

The classification and nomenclature of MIPs and WCPSs is an important issue in view of the ubiquity and continuous increase in numbers of MIPs (over 800) and WCPSs (over 450). The name aquaporin was proposed after the first WCPSs were cloned (CHIP28, WCH-CD, and γ–TIP), for “proteins related to CHIP, WCH-CD, and γ–TIP, which function as primary Water pores” and “should not be used to describe proteins permeated by ions (MIP26 conducts ions when reconstituted into planar lipid bilayers) or other molecules (GlpF is the glycerol facilitator of Escherichia coli)”4. However, as more and more MIPs and WCPSs have been discovered and their structure was deciphered the name aquaporin was extended to aquaglyceroporins and GlpFs, to “superaquaporins,” and to all structurally related proteins even of unknown functions (actually no difference between aquaporins and MIP proteins is made this way). Moreover, some authors are using the term aquaglyceroporin superfamily to include aquaporins, aquaglyceroporins and glycerol facilitators52; others are including these three groups of membrane proteins in the “aquaporin family”53, or “aquaporin superfamily”. In addition, while some authors are considering aquaglyceroporins and glycerol facilitators as equivalent54, 55, others propose the classification of MIP proteins into three functional subgroups: AQPs, glycerol-uptake facilitators and “aquaglyceroporins”56. Recently, aquaporins were classified into three “major subtypes, determined by their transport capabilities”: the “classical” aquaporins, aquaglyceroporins, and “unorthodox” aquaporins51.

In addition, aquaporin is abbreviated by various authors either as AQP or as AQP and glycerol facilitator either as GlpF55 or GLP54. I propose to use the abbreviations AQP and GlpF.

More comprehensive classifications of MIPs, based on the primary sequences and suggesting evolutional pathways, are available (53,56–58).

STRUCTURAL CHARACTERISTICS OF WCPSs (AND OF MIPs)Several characteristic structural features of WCPSs (and of MIPs) were described (53, 59–63). WCPSs and MIPs

have a relatively small size: most are less than 300 amino acids in length, usually 250–290. Both the NH2 terminus and the

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COOH terminus are hydrophilic and located in the cytosol. In the amino acid sequence there are two highly conserved regions called NPA boxes (or motifs) with three amino acid residues (asparagine, proline, alanine: Asn-Pro-Ala) and several surrounding amino acids. The NPA boxes have been called the “signature” of WCPSs. However, the analysis of MIP family database revealed considerable variation of NPA motif [reviewed in54, 56]. WCPSs and MIPs have considerable similar sequences of amino acid residues in the first and the second halves of the polypeptide chain (i.e., there are two tandem sequence repeats), as first noticed by Wistow et al.61. The two repeats probably evolved by gene duplication55.There are six transmembrane domains (TMDs), highly hydrophobic, with α-helix structure and five connecting loops. The α-helices are named from the N-end succesively H1, H2, H3, H4, H5, and H6, and the five loops are named A, B, C, D, and E (Fig. 1). The TMDs and the loops form a core60 (embedded in the membrane lipid bilayer), to which two “legs” (represented by the cytosolic N- and C-ends) are attached. The NPA boxes are located in the loops B and E, which are rather hydrophobic in nature and have short (half) helices HB and HE. The six TMDs (tilted at about 30° with respect to the membrane normal) form a right-handed bundle enclosing the channel (pore) formed by the NPA motifs and the short tetramer helices HB and HE, bended into the six-helix bundle and connected in the center of the bilayer. This structure is called the aquaporin fold64. So the channel (pore) is a narrow tunnel in the center of the molecule, that has at the extracellular and cytoplasmic faces funnel-shaped openings (atria telpa or vestibules priekštelpa). This model was called “hourglass model”59. Figure 1. Various views of the prototypical aquaporin (AQP)1 crystal structure.(A) Cartoon

of an AQP1 monomer as viewed from the side depicting the two repeated protein halves (blue and yellow helices) and the two short pore forming helices HB (green) and HE (red). The connecting loops are shaded in gray. (B) Vertical cross-section of AQP1 showing the location of the conserved aromatic/arginine (ar/R) constriction and the Asn-Pro-Ala (NPA) region. The arrows indicate the viewing direction on (C) that is, residues of the ar/R constriction, and (D) the NPA region of AQP1. Modified by Dr. Binghua Wu (Dept. of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Univ. Kiel, Germany) after the original published in ref.84 and reproduced with permission from Birkhauser.

In the natural membranes or in model membranes (reconstituted proteoliposomes with purified proteins) WCPSs are in the form of tetramers, as shown by freeze

fracture electron microscopy62. AQP1 tetramers are held together by extensive interactions between helices and loops of the monomers. Each monomer, however, has its own channel, functionally independent63. Out of the four monomers only one or two are glycosylated. There is a single N-glycosylation site in the second extracellular loop (C). The nonglycosylated form has molecular weights of 26–30 kDa and the glycosylated form has molecular weights in the range of 35–60 kDa.

The structural features of WCPSs and MIPs became better understood after the atomic structure of some proteins of the superfamily was deciphered: human RBC AQP1 (hAQP1)64, 65, bovine AQP1 (bAQP1)66, E. coli GlpF67, 68, E. coli AQPZ69, 70, eye-lens specific AQP071,72, archaebacterial AQPM73, plant SoPIP2;174, AQP4, the predominant Water pore in brain75, and recently hAQP576. There are other MIPs currently known at low or intermediate resolution. In addition, much has been learned about structure and function of Water channels, particularly regarding the dynamics of Water permeation and the filter mechanism from molecular dynamic simulations of AQP1 and GlpF (68, 76–81).

The first atomic structure of a WCPS (and the first atomic structure of a human membrane protein) to be solved was that of hAQP1 from the RBC membrane at 3.8 Å resolution obtained by electron crystallography64. The refined structure of hAQP1 was deciphered after the structure of bAQP1 and of E. coli GlpF at 2.2 Å resolution were analyzed by X-ray crystallography66. the glycerol facilitators (abbreviated as GlpFs, from Glycerol permease Facilitators)

The structure of AQP1 and the details of the channel (pore) are given in Fig. 1.The dipoles of the short helices HB and HE, which form the channel together with the tightly stacked NPA motif s

create a functionally important electrostatic field at the membrane center (64, 68, 79). The surface of the AQP1 pore is formed by highly conserved residues in H2 and H5 and the C-terminal halves of the H1 and H4 (forming the remaining surface of the pore). six Water molecules form a single file through the pore66. The physical limitation on the size of substrates allowed to permeate the AQP1 pore is imposed by the 3 Å diameter of the narrowest region of the pore64, which is only slightly larger than the 2.8 Å diameter of the Water molecule . The pore constriction prevents permeation of all molecules bigger than Water , including hydrated ions. The narrowest region of the pore in AQP1 was named the Ar/R constriction site, because it contains highly conserved aromatic and arginine residues66. The Ar/R constriction site is formed in hAQP1 by Arg195(197), His180(182), Phe56(58), and Cys189(191)66. Arg 195(197) and His180(182) line one side of the pore creating a hydrophilic surface, whereas the Phe56(58) is located on the opposite side. Cys189(191) is the site for the inhibition by mercurials of Water permeation through the pore.

Despite its extreme Water permeability, allowing permeation of 3 × 109 Water molecules per monomer per second21, AQP1 (and other WCPSs) strictly prevents the conduction of protons. This is physiologically very important, as the passage of protons through the pore would anihilate the proton gradient across the cell membrane that serves as a major energy storage mechanism. The proton exclusion may be seen as the most exceptional feature of AQP(1)s, and the NPA motifs play an important role. The two asparagines at the positive ends of helices HB and HE act as hydrogen donors to the oxygen atom of the Water molecule in the center of the pore. The Water molecule is oriented perpendicular to the pore axis; the central Water molecule forms (by its oxygen) hydrogen bonds with the amido

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groups of Asn76(78) and Asn192(194); this Water molecule can only engage in hydrogen bonding leading outwards from the center of the pore toward the extracellular and the cytoplasmic entrance of the pore. The lines of Water molecules in the two pore halves thus have opposite hydrogen bond polarity, preventing protons to cross the central Water molecule78.

The electrostatic proton barrier in AQPs involves not only the NPA motif s , but also the Ar/R constriction size 79 . Mutation experiments showed that removal of the positive charge from the Ar/R constriction site in two AQP1 mutants, Arg195Val and His180Ala/Arg195Val, appeared to allow the passage of protons through the AQP1 pore80. The positive charges of an arginine residue at the extracellular vestibule and of histidine residues in the cytoplasmic vestibule would also help to repel protons from entering the pore 64 . In addition to these electrostatic factors another major source of the barrier for proton transport in AQPs is associated with the loss of the generalized solvation energy upon moving the proton charge from the bulk solvent to the center of the channel 81.

Yool et al.82 proposed a role for AQP1 as a cyclic nucleotide-gated cation channel (the channel being located in the midle of the tetramer); however, this suggestion was criticized 83.

On the other hand the CO2 permeability of AQP1 [(discussed in J. Physiol., 542, 2002 and in84] is controversial, even in recent publications, particularly in regard with its physiological significance (85-88). Transport of CO2 by some plant AQPs was reported 89, 90. In addition, evidence for passage of NO through the AQP1 was published 91. Other WCPSs are permeable for H2O2, ammonia NH3,antimonite,arsenite,silicic acid,O2,COhttp://aris.gusc.lv/BioThermodynamics/BiologicalBuffers.doc

The availability of high resolution structural data and molecular dynamics simulations made possible the description of the mechanisms of gating (i.e., the opening and closure of the pore) for some WCPSs. two mechanisms have been proposed: “capping” and “pinching” [see Fig. 5 in92]. “Capping” require a large-scale rearrangement of cytoplasmic loop to completely stop the Water passage through the pore. This occurs in case of channels with a very high Water conductance, such as plant SoPIP2;1. The atomic structure of SoPIP2;1 from spinach was determined in an open and closed pore state and the mechanism of gating has been described74. In the closed (unphosphorylated) form the cytoplasmic loop D (which is 4-5 residues longer in plant MIPs vs. mammalian ones), is held in its closing position through H-bonds with the N-end. Upon phosphorylation, the connection of the N-end and loop D is broken and the latter is free to undergo large conformational changes resulting in the opening of the Water pore by two complemantary mechanisms: a) displacement of loop D from the cytoplasmic mouth of the channel, and b) retraction of a hydrophobic, pore-lining residue from the pore74.

“Pinching” implies smaller movements of a few residues, or a single residue, which pinch in upon the Ar/R constriction region and thereby restrict the passage of Water. This occurs in case of channels displaying poor Water conductance, that is further decreased by gating. Such is the case of AQP0 with a measured Water permeability 15-fold lower than that of AQP1 at pH 6.5; this is reduced a further three fold at pH 7.5 [see citations in92]. It was proposed that differences in the packing of AQP0 induce a gating effect due to close contacts between the extracellular domains , which results in different conformations of extracellular loop A ; the movement of loop A slightly displaces Met176 and His40 into the channel in the putative closed conformation, and the Water pore of AQP0 becomes more constrained near the conserved Ar/R constriction site [see Figs. 3b and 4 in92].

In case of AQPZ molecular dynamics simulations 93 revealed that Arg189, which is a strictly conserved component of the Ar/R selectivity filter, flips between two distinct stable conformations: one in which the head group orientates “upwards” towards the extracellular medium and the Water channel is open; and one in which the head group orientates “downwards” into the pore and thereby closes the channel. Such a mechanism of gating received support from the X-ray structure of AQPZ 69, 70.

WCPSs (AND OTHER MIPs) IN UNICELLULAR ORGANISMS After the discovery of the first WCPS, called AQPZ, in E. coli44 a widespread existence of aquaporins among bacteria was suggested, with roles in measurement of osmotic stress, cell growth and division, or desiccation44, 94. These suggestions have not been confirmed. Although WCPSs have been identified in some bacteria and other unicellular organisms ranging from archaea to eukaryotes, aquaporins are not present in all species of microorganisms. When all sequenced microbial genomes were considered, 153 microbial species apparently devoid of aquaporins were identified, whereas putative aquaporin-encoding genes have been found in only 71 species; in addition, aquaporins seem to be more abundant in eukaryotes (67% of 33 species) than in prokaryotes (26% of 193 species) and are found in only three of the 22 sequenced archaea. Among the fungi, no aquaporins have been reported in the basidiomycetes, whereas they are present in many lower fungi47. The absence of WCPSs in many microorganisms indicates that aquaporins are probably not required for processes that are universally important for microbial survival, including cell growth and division. No clear common phenotypes caused by inactivation of aquaporins are apparent47. The lipid counterpart of the membrane is probably sufficiently permeable to Water, so that, given the high surface area to volume ratio of the microbial cell, a significant role of AQPZ in Water uptake in growth appears unlikely95.

Functional analyses are needed to confirm that all the currently identified putative aquaporins in microorganisms have a function in Water transport. To date, only the Water transport functions of AQPZ, of M. marburgensis AQPM and Saccharomyces cerevisiae AQY1 have been confirmed experimentally47.

The involvement of AQPZ and other microbial aquaporins in osmoregulation is discussed by Kayingo et al.48. Mutants of AQPZ have been used to study the structural basis of aquaporin inhibition by mercury96. It was concluded that inhibition of AQPs by mercury is due to the blockage of the pore by a steric mechanism and not by a conformational change of the protein.

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On the other hand microorganisms have a high number of GlpFs [reviewed in48]. In fact GlpFs account for the majority of MIPs in microorganisms47. The E. coli GlpF was first identified in 1960s and its permeability to glycerol, urea and glycine was established by 1980 [Heller et al. 1980, cited in71]. However, it was not recognized as a relative of WCPSs until the 1990s, probably because its Water permeability is much lower than that of AQPs71. E. coli GlpF shares a high amino-acid sequence similarity with AQPZ; however, it conducts Water at a rate that is only about one sixth that of AQPZ [Borgnia and Agre, 2001, cited in71].

The GlpF structure67, 68 revealed the aquaporin fold with six membrane-spanning helices and two half helices forming a right-handed bundle surrounding an aqueous channel (pore), so that GlpF was called the “fraternal twin” of AQP197 The constriction site in GlpF (larger than in AQP1 or AQPZ), has a diameter of 3.14 Å, which is large enough to accomodate a glycerol molecule67, and is also more hydrophobic than that in the pore of AQP1. A mutant GlpF, W24F/F200T, reconstituted in liposomes, showed reduced glycerol efflux rates while the Water efflux rate increased68 demonstrating the shifting of GlpF channel properties towards that of aquaporin98.

In regard with WCPSs in archaea the DNA sequence of such a protein, called AQPM, a candidate aquaporin or aquaglyceroporin, has been recognized in the genome of M. marburgensis, a methanogenic thermophilic archaeon99. AQPM was expressed in E. coli, then purified and reconstituted into proteoliposomes, where AQPM behaved like a moderate mercury sensitive Water channel and a very poor glycerol transporter45.

As the MIP superfamily was believed to date back 2.5–3 billion years in evolutionary time57, recognition of an aquaporin in an archaeon suggested an even earlier origin, although it is possible that the gene was transferred horizontally from other microorganisms [Salzberg et al. 2001, cited in45]. From phylogenetic analyses it was suggested45 that eukaryotic members of the MIP family evolved from two basal lineages: AQPZ-like Water channels and GlpF-like glycerol facilitators. These divergent lineages may have originated from an AQPM-like sequence, which appears to be intermediate in sequence between the Water-selective aquaporins and the aquaglyceroporins [Zardoya et al., 2002, cited in54].

The physiological role of aquaporins in archea is not known. It was suggested45 that in some way it should be related with the ability of archaea to withstand exceptional challenges in maintaining Water balance as they thrive in extreme environments including saturated salt solutions, extreme pH and temperatures up to 130°C.

The number of WCPSs (aquaporins plus aquaglyceroporins) in yeasts can range from one to five. Fungal aquaporins have been studied inS. cerevisiae and in C. albicans. There are two aquaporins in S. cerevisiae, called AQY1 and AQY2, and only one in C. albicans. AQY1 and AQY2 are highly similar (88% identical), indicating a recent gene duplication; however, the expression of the two aquaporin genes (AQY1and AQY2) is regulated differently, indicating functional specialization100. The inactivation of AQY1 and AQY2 in S. cerevisiae and deletion of the single aquaporin gene in C. albicans have no conspicuous effects on growth under a variety of conditions (47 and refs. therein). In addition, both S. cerevisiae and C. albicans aquaporin-deletion strains are more resistant to rapid changes in osmolarity compared with wild type. Consequently, a role for aquaporins in microbial osmoregulation (turgor regulation or osmoadaptation) seems improbable, because the Water channel activity would aggravate osmotic stress-induced problems rather than counteract them (47, 95).

WCPSs in microorganisms might play a role in Water transport during natural dehydration processes, for example, spore formation: the Water content of spores is aproximately half of that of vegetative cells100. Such a role has been demonstrated for AQY1 in the formation ofS. cerevisiae spores101. Mutants lacking AQY1 show decreased spore viability and this was related to events occuring during spore formation rather than during spore maintenance or germination102. However, subsequent studies102 indicated that S. cerevisiae wine strains lacking AQY1 did not show a decrease in spore fitness or enological aptitude under stressful conditions, limited nitrogen, or increased temperature (heat stress). The physiological role of AQY2 is less clear than for AQY1 [see100 for discussions].

A role for microbial aquaporins in sustaining low-temperature Water permeability, and in this way providing protection of cells against freeze-thaw stress, has been suggested by Tanghe et al. (47, 103). Such a protective effect of aquaporin overexpression has been found in S. cerevisiae, C. albicans, and S. pombe (47 and refs. therein). Some conditions in which WCPSs enhance microbial tolerance against rapid freezing and thus the presence of aquaporins might be advantageous for survival include the following: the liberation and subsequent freeze-thawing of microorganisms by the activities of warm-blooded organisms in frozen environments (breathing, sneezing, flying, stepping and running on frozen ground, contact with trees and other obstacles, foraging on frozen plants, the excretion of feces and urine), large-scale air dispersal of microorganisms associated with dust particles in clouds that can travel between continents, or the “freezing rain”47.

Yeasts have a range of genes that encode aquaglyceroporins, those from S. cerevisiae (Fps1 and Yfl054) being best studied100. Fps1 was in fact one of the first WCPSs discovered104. As discussed in an excellent review by Hohmann105, Fps1 plays a role in the osmoregulation of S. cerevisiae, controlling the intracellular level of glycerol, the compatible osmolyte of proliferating yeasts cells.

Fps1-like aquaglyceroporins have been found in other yeasts and a comparison of their structure is discussed by Petterson et al.100. Briefly, the predicted proteins differ in length by as much as 216 residues, as there are several alterations in the NPA motifs.

S. cerevisiae Yfl054 and Yfl054-like aquaglyceroporins are characterized by an approx. 350 amino-acid long N-terminal extension and an approx. 50 amino-acid long C-terminal extension. The physiological role of these aquaglyceroporins is currently unknown100.

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In conclusion, although WCPSs have been found in bacteria, archaea, and unicellular eukaryotes, their absence in many microorganisms appears to indicate that rather than fulfilling a broad role such as osmoregulation, instead WCPSs are involved in specific processes (such as sporulation) or improve freeze tolerance under rapid-freezing conditions; this WCPS function might be important for survival at low temperatures. Future studies are necessary to evaluate other possible physiological roles of WCPSs in microorganisms47, 100.

WCPSs in protozoa have not only physiological importance, but also relevance for tropical parasitic diseases. A role in sporulation and in spore germination has been suggested for the putative Water channel WacA in an amoeba, Dictyostelium discoideum106. The role of Water channels in the developmental process of D. discoideum was further investigated by Mitra et al.107, who identified another WCPS called AQPA and sugessted that it is essential to maintain spore dormancy perhaps through the regulation of Water flow.

An AQP gene from Amoeba proteus was recently cloned108. The protein, called ApAQP is a specific Water channel present in the membrane of the contractile vacuole (CV) and was implicated in the Water transfer across the CV membrane. In freshWater protozoa Water enters the cell along the osmotic gradient across the plasma membrane since the osmolality of the cytoplasm is higher than that of the environment. The CV periodically repeats a cycle of slow expansion to fill with the fluid from the cytoplasm (diastole) and quick contraction to release the fluid to the cell exterior (systole) [Patterson, 1980, cited in108].

WCPSs from pathogenic protozoan parasites are presented in excellent reviews by Beitz109, 110. Among these parasites are the organisms of the phylum Apicomplexa: Plasmodium species (the causative agents of malaria), Toxoplasma gondii (toxoplasmosis), as well as those of the order Kinetoplastida: Trypanosoma brucei (sleeping sickness), Trypanosoma cruzi (Chagas'disease) andLeishmania species (leishmaniasis). The majority of the Apicomplexa have a single aquaglyceroporin gene, while up to five such genes have been identified in the genomes of the Kinetoplastida. The proteins encoded by these genes appear to be mostly aquaglyceroporins. They have been proposed to play physiological roles in the protection of the parasites from osmotic stress during kidney passages or during transmission between human and insect as well as in glycerol uptake as a precursor for membrane lipid biosynthesis110.

On the other hand the protozoan WCPSs appear to be potentially important for use as a target or entry pathway for chemotherapeutic compounds109.

Inspection of the completed genomes of protozoa provided a major surprise. The Apicomplexan Cryptosporidium parvum does not have any WCPS gene. This is the first identification of a eukaryotic organism that totally lacks WCPSs109.

WCPSs (AND OTHER MIPs) IN PLANTS Since the first identifications of WCPSs in plants42, 43 tremendous progress in the knowledge of plant WCPSs has been achieved, regarding their occurrence, structure, permeability characteristics, regulation, and their physiological roles. In addition to WCPSs, plants have MIPs which transport other substances. Plant MIPs are more abundant and show greater diversity than those in bacteria or animals, as MIPs are required for multiple functions (111-117): (a) plants can mediate a large flow of transpiration: soil Water is absorbed by the root, moves radially through living cells to reach the vascular tissues (xylem vessels), by which is transported in shoots. Water is eventually lost in the atmosphere by transpiration, through the stomatal pores of the leaves (on a warm, dry day, the leaf may exchange the equivalent of all its Water content in 20–60 min; (b) plants have to achieve a three-dimensional control of Water exchange in living tissues; (c) this control has to be exerted during all stages of plant growth and development and has to respond to various environmental conditions; (d) plant cells are highly vacuolated and this requires tightly coordinated control of Water and solute transport across the plasma membrane and the intracellular membranes. WCPS-mediated Water transport seems also to be crucial during leaf and petal movements, reproduction, cell elongation, and seed germination. A role for intracellular WCPSs in plant cell osmoregulation has been proposed (113, 114, 117). In addition, a role of a tobacco aquaporin in CO2 uptake during photosynthesis has been documented90.

It is thus understandable why a high abundance and multitude of MIPs have been identified in plants and it seems that all plants have such proteins. A remarkable multiplicity of MIP isoforms has been identified by genome sequencing: 35 in Arabidopsis thaliana, 33 in rice, 36 in maize116. The plant MIPs are found both in plasma membranes and in intracellular membranes and can be subdivided in four groups (or subfamilies), which to some extent correspond to distinct sub-cellular localizations (113, 115, 116): (a) the tonoplast intrinsic proteins (TIPs) abundantly expressed in the vacuolar membranes; there are various isoforms of TIPs: alpha (seed), gamma (root), and Water-stress induced (Wsi)117; (b) the plasma membrane intrinsic proteins (PIPs), located in plasma membranes, can be subdivided into two phylogenetic subgroups PIP1 and PIP2; (c) the Noduline 26–like intrinsic membrane proteins (NIPs), that is AQPs that are closed omologues of Gm Nod26, an abundant AQP in the peribacteroid membrane of the symbiotic nitrogen–fixing nodules formed after infection of soybean by Rhizobiaceae bacteria118; (d) the small basic intrinsic proteins (SIPs), first uncovered from genome sequence analysis119, form a class of 1–3 divergent aquaporin homologues and are located in the ER (endoplasmic reticulum) membrane120.

Novel types of MIPs have been recently described: a homologue of the bacterial GlpF, acquired by the moss Physcomitrella patens by horizontal gene transfer121, and a fifth class of plant MIPs, which are closely related but yet clearly distinct from PIPs, found in the moss and poplars122.

TIPs promote the transport of Water and small uncharged molecules across the vacuolar membrane (tonoplast). Some TIPs are highly selective AQPs, ensuring a 100-fold higher Water permeability of this membrane compared to plasma membrane122; such TIPs may permit rapid osmotic equilibration between the cytosol and the vacuolar lumen, buffering the osmotic fluctuations of the cytosol, and regulating cell turgor (113, 114, 117). Other TIPs are permeable to Water, urea and glycerol123 and such WCPSs may play a role in equilibrating urea concentrations between the vacuole

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and the cytoplasm. In addition, the participation of TIPs to transport ammonium (NH ) and its gaseous conjugated base (NH3, ammonia) from the cytosol into the vacuole was suggested. NH /NH3 are additional N sources and primary substrates for the synthesis of amino acids. Consequently, TIPs appear to be involved not only in osmoregulation but may be linked to important metabolic pathways like the urea cycle or amino acid synthesis113.

PIPs, which form the largest plant MIP subfamily, are the central pathways for transcellular and intracellular Water transport (111-117). Although the amino acid residues at the selectivity filters are similar in PIP1 and PIP2 their permeability and cellular functions are different113. PIP1s could be transporters for small solutes and gases (CO2) in leaves and they need to be activated in roots in order to function as Water channels. PIP2s seem to be more efficient Water channels than PIP1s, and may provide a major pathway for cellular Water transport in roots, leaves, reproductive organs and seeds; in addition, CO2 transport mediated by PIP2s was proposed (113 and refs. therein).

NIPs are permeable to Water, glycerol, formamide and possibly to gaseous NH3. NIPs are also present in nonleguminous plants, where they have been localized in plasma and intracellular membranes116. NIPs can also be subdivided in two subgroups116. Members of the first subgroup (NIP I) are very comparable to the Gm Nod26 archetype and behave like aquaglyceroporins118. Solute transport mediated by NIPs may play a role in osmoregulation of the peribacteroid space. Members of the NIP II subgroup have a predicted pore of higher size than in the NIP I subgroup and, as a consequence, are permeable to larger solutes such as urea. However, they have a reduced Water permeability for unknown reasons. Recently, very original functions of members of the NIP II subgroup were identified: the uptake of boron by roots124 and the silicon uptake and transport throughout the plant125.

The SIPs subfamily is represented by three members in Arabidopsis (SIP1;1, SIP1;2, and SIP2;1) and in maize and by two members in rice. SIPs have several structural characteristics [reviewed in126]. (a) The first NPA motifs are changed to NPT, NPC and NPL in SIP1;1, SIP1;2 and SIP2;1, respectively. (b) The N-end is shorter than in other plant MIPs and it is posible that the N-end is related to the intracellular destination. (c) SIPs are relatively rich in basic residues (such as lysine). (d) The loop C between TMD3 and TMD4 is shorter (14–19 residues) than that of PIPs and TIPs (22–26 residues) and this might affect the tertiary structure of SIPs.

All SIPs have been demonstrated to be localized in the ER membrane, although it is not clear which is the ER retention signal [reviewed in126]. Characteristic expression patterns of each SIP has been found; for example SIP1;1 was detected in high amounts in young roots and flower buds, while SIP2;1 was accumulated in young roots and open flowers126. It was suggested that each SIP may play cell-specific roles. However, such roles are not clear. SIP1;1 and SIP2;1 have been demonstrated to have Water channel activity, while the transport substrate for SIP2;1 remains to be determined. It is not clear whether SIPs have a role in the specific functions of the ER or in maintaining the tubular/Reticular/sheet shapes of the ER. The nearest homologues of SIPs in mammalians are AQP11 and AQP12, as pointed by Ishibashi49. Further studies are needed to define the physiological roles of SIPs in ER membrane.

There are multiple mechanisms by which plant WCPS activity is changed and regulated. two aspects may be considered: a direct and probably rapid control of the channel activity by gating and a more complex regulation, including changes in gene expression, in trafficking of MIPs, in various physiological conditions or under environmental stresses (92, 113–117, 127). Factors affecting directly the gating include the following: phosphorylation, heterotetramerization, pH, cations, pressure, solute gradients, temperature. In addition, the permeability of WCPSs is influenced by hormones, nutrient stress, plant hormones, attack by pathogens. The phosphorylation sites are located in the N-terminal and C-terminal segments and also in loop B. Calcium dependent protein kinases are involved in phosphorylation that results in the pore opening112, 116. Cytosolic proteins decrease the Water permeability of PIPs (and TIPs). A coordinated inhibition of PIPs and, as a consequence, a general block of root Water transport during anoxic stress (resulting from soil flooding) was attributed to closure of the channel after cell acidosis128.

In addition to the rapid inhibition of root Water permeability by gating (closure of Water channels induced by the acid cytosolic pH) a general downregulation of PIP and TIP genes occurs in response to hypoxia. There are many other changes of MIPs in response to a variable environment. Light116 induces the diurnal regulation of the abundance of TIP in guard cells (controlling the stomatal aperture), or of a PIP2 in the pulvinus, the motor organ controlling the movement of leaves and leaflets in the Mimosaceae. Diurnal variations of root Water transport (with a twofold to threefold increase during the day) have been observed in many plant species and are matched or slightly preceded by an increase in the abundance of PIP1 and PIP2 transcripts. However, the abundance of the PIP proteins in roots shows a more complex diurnal variation profile, suggesting a role for posttranscriptional regulation129.

The regulation of MIPs under the effects of Water and nutrient stress is a complex issue, because the expression of different MIP genes may be stimulated, reduced, or unchanged under abiotic stress117. In roots of most plant species investigated, drought or salt stresses result in a marked decrease of Water permeability, probably due to the downregulation of Water channel activity (downregulation of most WCPS transcripts) during the day; this early response may provide a hydraulic signal to the leaf to trigger stomatal closure, whereas during the night, it may avoid a backflow of Water to the drying soil116. A recovery toward initial transcript abundance occurs over longer term stresses [reviewed in (116-117)].

Adaptation to salinity implies two phases. In the first phase (over the first 24 h of stress) a coordinated transcriptional downregulation and subcellular relocalization of both PIPs and TIPs in roots occur (with some transcripts downregulated during the first 15–60 min of salt exposure). After 7 days the expression level of transcripts recovered, and even became higher than in plants grown in standard conditions [discussed in (116-117)].

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Chilling of plants (i.e., exposure to 4–8°C) induce complex responses involving WCPSs: a marked decrease in root Water transport (associated with a twofold to fourfold decrease in the abundance of most PIP transcripts), closure of petals (for instance in tulip), associated with decreased phosphorylation of the putative WCPS [reviewed in116].

Biotic interactions of plants with soil microorganisms are important in plant Water relations and tolerance to environmental stress. NIPs behave like aquaglyceroporins and when phosphorylated their Water and solute permeability increased, while gas permeability decreased. Phosphorylation of Gm Nod26 (by calcium-signaling pathways) was enhanced in response to osmotic stress (both drought and salt stress).

In conclusion, WCPSs (and other MIPs) play a key role in plant Water relations. Novel functions and regulation mechanisms of plant WCPSs have been recently uncovered. Besides Water some plant MIPs can transport physiological important molecules such as neutral solutes (urea, boric acid, silicic acid) or gases (NH3, CO2). Thus plant MIPs are involved in many great functions of plants, including nutrient aquisition, carbon fixation, cell signaling, and stress responses (111, 117, 123).

WCPSs (AND OTHER MIPs) IN SOME MULTICELLULAR ANIMAL SPECIES WPCs have been discovered in animals at all levels of life, as well as in almost all organs and tissues of humans and a variety of roles have been documented or suggested. Selected examples are described below.

A genome project focusing on the nematode Caenorhabditis elegans has revealed the presence of eight MIP-encoding genes58. A functional analysis of this gene family has been performed by cloning the cDNAs for all eight genes and expressing them in Xenopusoocytes130. It was found that the genes named by the authors as AQP-2, AQP-3, AQP-4, AQP-6, and AQP-7 are encoding aquaporins, the genes AQP-1, AQP-3, and AQP-7 are encoding aquaglyceroporins, while expression of AQP-5 or AQP-8 had no effect on oocyte glycerol, Water, or urea permeability (i.e., these genes are encoding MIPs with no identified channel functions). The AQP genes were found to be expressed in several cell types, including the intestine, excretory cell, and hypodermis. Deletion alleles for AQP-2, AQP-3, AQP-4, and AQP-8 were isolated. Single, double, triple, and quadruple AQP mutant animals exhibited normal survival, development, growth, fertility, and movement under normal and hypertonic culture conditions. The quadruple mutants exhibited a slight defect in recovery from hypotonic stress but survived hypotonic stress as well as wild-type animals. The authors concluded that C. elegans MIPs are not essential for whole animal osmoregulation and/or that deletion of MIP genes activates mechanisms that compensate for loss of Water channel function130.

The first insect MIP, called Big Brain (BIB) protein has been located in the brain of Drosophila melanogaster and seemed to play an essential role in embryonic development131. However, no Water channel function has been found for BIB.

The first insect AQP was discovered in 1995132 in the membranes of epitelial cells in the filter-chamber of Cicadella viridis (Homoptera) and was called AQPcic133. The filter-chamber is a highly differentiated part of the insect digestive tract where the excess of sap dietary Water is rapidly transferred along an osmotic gradient across the epithelial cells, from the initial midgut to the terminal midgut and the proximal part of malpighian tubules (MT)133. Water is eliminated through the hindgut. The insect MT are the primary excretory and osmoregulatory organs, analogous to the vertebrate renal tubule [see134 for a short description). When expressed in Xenopus oocyte system AQPcic appeared to be a Water channel similar to AQP1 under the same condition, in regard with the permeability value and specificity, although it had a sequence that was only 43% identical to AQP1.

In parallel, a first Drosophila AQP (called DRIP, from Drosophila Integral Protein) was cloned from an adult Drosophila MT cDNA library135. The sequence of DRIP is the most similar to human AQP4. DRIP was found to be a Water specific channel, expressed at many stages during development, from embryonic to adult MTs136. Seven additional Drosophila MIPs have been discovered; however, their function as AQPs has not been determined136. Drosophila AQPs have been proposed to maintain fluid homeostasis, which is particularly important because flies are at constant risk of dehydration as a result of their high surface area-to-volume ratio [O'Donnell and Maddrell, 1995, cited in136]. In addition, Drosophila undergo significant morphological changes during metamorphosis, so their fluid needs change considerably136.

DRIP is most closely related to putative AQPs cloned from the yelow fever carrier Aedes aegypti and the malaria carrier Anopheles gambiae; a detailed characterization of DRIP and related AQPs may provide one means to fight the spread of disease136. An AQP from A. aegipti, called AeaAQP, was found in the tracheolar cells associated with the MT; the function of this AQP may be the removal of Water from the inside of the tracheoles to facilitate oxygen supply to the highly active epithelium of the MT: the details of these physiological implications are discussed in137, 138.

Hagfish (Eptatretus burgeri) are agnathoans and are the earliest living marine craniates, that diverged from the main vertebrate lineage over 500 million years ago139. They differ physiologically from other marine vertebrates because they maintain an osmotic equilibrium with the surrounding sea Water (SW), mainly by retaining extracellular concentrations of NaCl in their body fluids nearly isoosmotic to the sea level; they have neither a passive net Water influx, like freshWater (FW) teleosts do, nor a net efflux, such as marine teleosts have. The gills are responsible for the NaCl concentration in the plasma; in the gill pavement cells a WCPS called EbAQP4 has been identified, that was considered to be the ancestral WCPS of mammalian AQP4, playing a role in basolateral Water transport in the gill pavement cells139.

Aquatic vertebrates have a potentially greater difficulty compared to terestrial vertebrates in maintaining osmotic homeostasis because they are surrounded by an external environment which is almost always in dis-equilibrium with their body fluids. For example, teleost fishhave distinct mechanisms to adapt and survive the osmotic challenges posed by the SW (hyperosmotic) or FW (hypoosmotic) environments140. The gill (the branhial epithelium) is the major site of Water

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exchange between the internal milieu and the external aquatic environment inhabited by teleost fish, being responsible for over 90% of the total body Water influx in FW [Motais et al., 1969, cited in140]. The first WCPS in teleost fish was identified by RT-PCR from the gill of the European eel (Anguilla anguilla) by Cutler and Cramb140and proved to be an AQP3 homologue; it was also found in the eye, esophagus, intestine [Lignot et al., 2002, cited in141]. SW acclimation resulted in a reduction of ∼65% in the expression of branchial AQP3. Subsequently, similar studies have been performed in the other fishes by various authors [reviewed in141] suggesting that AQP3 plays a complex role within the major osmoregulatory organs of teleost fish.

In addition to AQP3 other WCPSs have been described in teleost fish, three of them being homologues of AQP1; these, plus a novel WCPS called sbAQP (from sea bram, Sparus auratus) and AQP3 have been reported to be expressed within the gastrointestinal tract (being implicated in the intestinal Water absorption), in the gill, kidney, and lens [Santos et al., 2004, Aoki et al., 2003, Virkki et al., 2001, cited in142].

AQP1 and AQPe (an aquaglyceroporin) isoforms from the European eel shared 35-54% identity with other known human AQPs; SW acclimation induced a marked increase in abundance of AQP1 in the intestine; as SW acclimation is associated with increases in drinking and Water uptake across the gastrointestinal tract, a role for AQPs in Water absorption in the teleost intestine is a distinct possibility142.

An AQP is involved in an unique physiological process among lower vertebrates (including the teleost): the oocyte hydration during meiosis resumption (or oocyte maturation). This rapid Water uptake contributes to the positive buoyancy of fish eggs and early embrios in the ocean which is essential for their survival and dispersal. A novel AQP, called SaAQP1o, related to mammalian AQP1, was identified in the ovary and in the oocyte of the gilthead sea brahm (Sparus aurata)143.

Amphibians are the first vertebrates that emerged from aquatic habitats to terrestrial environments. Many adult anurans have specialized organs to adapt to dryer environments (to compensate the strong evaporative loss when the animal is on land): the ventral pelvic patch (seat patch), to absorb Water from the external environments, and urinary bladder to store Water and reabsorb it in times of need [144 and refs. therein]; the urinary bladder, appearing for the first time in vertebrates.

The first amphibian WCPS was isolated in 1994 from frog (Rana esculenta) urinary bladder, a model for the kidney collecting duct145. The protein, called initially FA-CHIP (frog aquaporin-CHIP), showed 74% identity with hAQP1 and its Water channel function was characterized with the Xenopus levis oocyte expression system. In subsequent work146 FA-CHIP mRNA was found to be abundantly expressed in the frog urinary bladder, skin, lung and gall bladder. Salt acclimation for 1–5 days markedly increased skin and urinary bladder FA-CHIP mRNA expression. It was concluded that FA-CHIP is a a homologue of hAQP1, specific for amphibian, permanently present in basolateral membranes with its own regulation at the level of transcription and is not the vasopressin sensitive Water channel146.

The anuran WCPSs have been recently reviewed144, 147; 17 distinct full sequences of AQPmRNA have been identified. A phylogenetic analysis suggested that anuran AQPs can be assigned to 6 clusters: types 1, 2, 3, and 5 are homologues of mammalian AQP1, 2, 3, and 5, whereas two are anuran-specific types: AQPa1 and AQPa2 (the letter “a” denotes anuran). AQP1 might play a role in the transport of absorbed Water into the blood stream through the capillaries located subcutaneously or in the bladder submucosa. AQP2 and AQPa2 types are the ADH-regulated Water channels that are translocated from the cytoplasmic pool to the apical plasma membrane of the granular cells of the pelvic patch and urinary bladder.

Of special note, in anurans AQPs are important in mediating physiologic responses to changes in external environment, including those that occur during metamorphosis and adaptation from an aquatic to terestrial environment and thermal acclimation in anticipation of freezing [147 and refs. therein].

WATER PERMEABILITY OF RBC IN VARIOUS SPECIES Although AQP1 was first discovered in the membrane of human erythrocytes (5, 6, 20), the physiological role of WCPSs in RBC was not clear for a long time. Smith et al.148 suggested a role in promoting the rehydration of the RBC after their shrinkage in the hypertonic environment of the renal medulla. When the cell is subject to shrinkage the hemoglobin is concentrated and the shrunken-hydrated cell is subject to blockage in the vasa recta, depending on the extent of the shrinkage95. The presence of AQP1 in the membrane serves to make this potential problem worse by augmenting the osmotic flows across the membrane that shrink the cell 95 .

Kuchel and Benga149, 150 provided new explanations for the physiological “raison d'etre” of AQPs in RBC. The first explanation is related to the membrane undulations (or oscillations) of the human RBC membrane, a phenomenon also called “flickering”151, 152. We formulated an “oscillating sieve” hypothesis: the high Water permeability of RBC membrane favors the energy driven membrane undulations (that perform a valuable role in movement of the cells through capillaries) such as these movements consume a minimum of energy in simply displacing Water. The second explanation (the “Water displacement” hypothesis) is: the high Water permeability of RBC membrane allows concomitant displacement of Water molecules when rapid entry and exit of ions such as Cl− and HCO , and solutes, such as glucose (all of whose molecular volumes are significantly greater than that of Water) is taking place.

Although the two explanations relate to molecular- and cellular-scale events another level of explanation is the correlation of RBC Water permeability with physical activity of the species. We have measured the membrane diffusional permeability to Water (Pd), and the activation energy (Ea,d) of this process in RBCs of over 30 vertebrate species (153-163). The parameters characterizing the Water permeability of RBC appear to be a species characteristic; there are no changes correlated with the marked alteration in the habitat of the species introduced to Australia (rat, rabbit, sheep, chicken) compared with their European counterpart. The chicken and echidna RBCs have the lowest Pd values (∼2

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× 10-3 cm s−1) and the highest values of Ea,d (over 30 kJ mol−1); this indicates that no functional AQPs are present in chicken and echidna RBCs. Human RBCs have Pd values of ∼4 × 10-3 cm s−1 at 25°C and ∼7 × 10−3 cm s-1 at 37°C with a value of Ea,d of ∼25 kJ mol−1. Large and less-active animals (cow, sheep, horse, and elephant) have lower values of Pd. In contrast, small and active animals (mouse, rat, guinea pig, rabbit, small marsupials) have Pd values significantly higher with lower Ea,d values (from 15 to 22 kJ mol−1). It appears therefore that AQPs in the RBCs ensure the rate of exchange of Water across the membrane required in various animals in relation to their physical activity, metabolic rate and the mean rate of circulation of their blood.

Studies on RBC Water permeability enabled our group to report other new facts. A positive correlation between the Pd values of RBCs from maternal venous blood and fetal RBCs isolated from cord blood taken at delivery was found. This points to a genetic basis for the determination of RBC Water permeability164.

In addition, we also have a priority in the discovery of the implications of WCPSs in human diseases. Benga and Morariu165 reported a decreased Water permeability of RBCs in children with idiopatic epilepsy (cases selected by Ileana Benga). On the other hand we found a decreased Water permeability of RBCs in Duchenne muscular dystrophy (DMD) patients166, 167. These findings were interpreted as an expression of generalized membrane defects affecting Water permeability in epilepsy and DMD. Our conclusion was confirmed by reports of implications of WCPSs in epilepsy 168 and DMD169.WCPSs IN HUMANS AND OTHER MAMMALS: FROM PHYSIOLOGY TO CELL AND MOLECULAR MEDICINE So far, 13 WCPSs have been discovered in mammals. Most authors consider that out of these, seven are aquaporins (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8), four are aquaglyceroporins (AQP3, AQP7, AQP9, and AQP10), whereas AQP11 and AQP12 are “superaquaporins” or subcellular AQPs. Recently, AQP6, AQP8, AQP11, and AQP12 were named “unorthodox” AQPs. The characteristics, distribution, functions and some pathological implications of individual mammalians WCPSs are presented in170, 171. In addition, the structure-function relationships in some organs, where several WCPSs occur, are briefly presented below and references of detailed reviews on each topic are given.

The kidney is the primary organ that regulates the Water balance and in which the WCPS functions are most well understood. Each kidney contains approximately one million nephrons and eight WCPSs are distributed specifically in the different segments of the nephron that have distinct roles in processing a blood filtrate to generate urine171–173.

AQP1 is abundant in the apical and basolateral membranes of epithelial cells in the proximal tubule and descending thin limb of Henle's loop (DTLH), and in the microvascular endothelium of outer medulary descending vasa recta (DVR). AQP7 and AQP8 are also present in the proximal tubule epithelium. These WCPSs are involved in the concentration of urine taking place in the proximal nephron (∼75% of the blood filtrate which is ∼150–180 L per day)171. The functional role of AQP1 in kidney was confirmed by investigations on mice and humans. Mice with a targeted knockout of AQP1 gene had polyuria and decreased urine–concentrating ability, as well as a decreased Water permeability of the proximal tubule and the descendingvasa recta [Ma et al. 1998, cited in171, 173. When deprived of Water for 36 hAQP1 null mice became profoundly dehydrated and serum osmolality markedly increased.

Colton (Co) blood group antigens corresponds to an alanine–valine polymorphism at residue 45 which resides in an extracellular location of AQP1 [Smith et al., 1994, cited by171]. Seven Colton-null families have been identified worldwide and individuals in three of these families, homozygous for distinct mutations in the AQP1 gene, had a complete absence or marked deficiency of AQP1. Surprisingly, these individuals suffered no obvious clinical consequences and had normal urine volumes. When deprived of Water the AQP1–null individuals had normal increases in serum osmolality and vasopressin levels; however, they had a limited ability to concentrate urine. This indicates that, in contrast to AQP-null mice, in AQP1-null humans, the primary defect is not in the proximal tubule, but rather in the DTLH and/or the DVR. As the manifestations of an AQP1 deficiency in mice are more severe than in humans, this indicates significant species-specific differences in the mechanisms of proximal-tubule Water reabsorption, or that AQP1-null humans have some form of compensation171.

Several WCPSs are expressed in the renal collecting duct (RCD) which determines the final volume and concentration of urine. AQP2 is present in the principal cells of the RCD and is located in the apical membrane and in the intracellular subapical vesicles. AQP2 is involved in the vasopressin regulated urine concentration (∼25% of the blood filtrate). Vasopressin is the ADH released from the pituitary gland that binds to its receptor on RCD epithelial cells inducing the translocation of vesicles that contain AQP2 to the apical membrane, which markedly increases RCD Water permeability; these findings confirmed the “shuttle-hypothesis” of Wade39. Water enters in the principal cell through AQP2 and exits through AQP3 (or AQP4) located in the basolateral membranes of principal cells. The mechanisms of the AQP2 trafficking have been extensively investigated [reviewed in (170-172, 174)]. Following the binding of ADH to its type 2 (V2) receptor, the synthesis of cAMP by adenylate cyclase is stimulated by a receptor coupled heterotrimeric GTP-binding protein. The increased cAMP levels activates protein kinase A, which phosphorylates AQP2 on Ser256 [Fushimi et al., 1997; Katsura et al., 1997, cited in171]. Vesicles containing AQP2 then undergo microtubule-mediated translocation to the apical membrane, where specific vesicle-docking proteins participate in the membrane fusion [Marples et al., 1998, cited in171].

Inherited defects in AQP2 cause 10% of cases of the very rare but very severe hereditary nephrogenic diabetes insipidus (NDI), one of the first examples of a clinically important WCPS defect175, a disease in which large volumes of dilute urine are excreted. (Approximately 90% of the cases of NDI are of X-linked recessive inheritance associated with the mutations in the vasopressin V2 receptor gene).The autosomal-dominant mutations in AQP2 produce trafficking defects: heterotetramers (composed of mutant and normal AQP2 monomers) are not transported to the plasma

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membrane. The autosomal-recessive mutations lead to the misfolding of the mutant monomers, which are presumably degraded and therefore cannot oligomerize with the normal monomers171. To date, approximately 35 mutations of theAQP2 gene have been reported, 27 of which are autosomal recessive176. All mutations found in dominant NDI are located in the AQP2 C-end, while nearly all mutations found in recessive NDI are located in the core region60. Mice models of NDI have been generated, and they are ideal models to develop for therapies for congenital NDI, as illustrated by the data of Sohara et al.177 indicating that dominant NDI might be relieved by treatment with rolipram, a phosphodiesterase four inhibitor.

Acquired NDI, because of decreased AQP2 synthesis, is common and has been observed in animal models: rats exposed to lithium, transient bilateral ureteral obstruction or chronic hypokalaemia or hypercalciuria. In contrast, excessively high levels of AQP2 have been described in conditions of fluid retention, including congestive heart failure178, cirrhosis179, pregnancy [171 and refs. therein].

Mice lacking both AQP3 and AQP4 have an even greater increase in urine production compared to AQP3-null mice, indicating a compensatory role of AQP4 in AQP3-null mice173.

AQP6 was found180 in the intracellular vesicles of acid-secreting intercalated cells of the RCD colocalized with the H+-ATPase, a protein that participates in the secretion of acid in the urine. AQP6 seemed to be a Hg2+-inhibitable Water channel [Ma et al.,1993, cited in51]. Later, it was found that AQP6 is also permeated by anions and channel function is activated by Hg2+ and low pH [Yasui et al., 1999, cited in171]. Lui et al., 2005 [cited in51] found that, in contrast to previous studies, AQP6 does not allow Water to permeate, but that a mutation of a single residue (a change of Asn-60 to Gly converts AQP6 from an anion channel to a Water channel. The majority of studies would characterize AQP6 as a unique, Hg2+-and low pH-activated, multipermeable channel. AQP6 was recently included among the “unorthodox” AQPs51. It was speculated that AQP6 may play a role in the body acid–base homeostasis171.

AQP7 [that was originally discovered in seminiferous tubules by Ishibashi et al.181] was found in the kidney colocalized with AQP1 in the brush border of the proximal tubule [Nejsum et al., 2000; Ishibashi et al., 2000, cited in51]. The main physiological role of AQP7 in the kidney is related to glycerol reabsorption51. AQP7-KO mice show greatly increased glycerol excretion182.

AQP8 is localized intracellularly in proximal tubules and RCD, however its cellular localization (in mitochondria) and regulation of targeting are topics of dispute [see discussions in51]. AQP8 can be permeated by NH3; however this may not be physiologically relevant because the AQP8 disruption did not lead to changes in NH3 metabolism [Yang et al., 2006, cited in51]. AQP8 was recently included among the “unorthodox” AQPs51.

AQP11 was found in the cytoplasm of the proximal tubule cells and, although its exact function is not known, the deletion of AQP11 gene produces a severe phenotype: AQP11-KO mice dye with polycystic kidneys following vacuolization of the proximal tubule183.

The gastrointestinal system accomplishes (next to kidney) the second largest amount of total fluid absorption and secretion. Substantial quantities of fluid are secreted into the gastrointestinal tract by salivary gland, stomach, pancreas and the hepatobiliary tree and the majority of secreted fluid is absorbed by the small intestine and colon to produce a dehydrated feces173.

The salivary gland expresses several aquaporins including AQP1 in microvascular endothelia, AQP4 in ductal epithelia, and AQP5 at the apical membrane of serous acinar cells. It seems that only AQP5 has a role in saliva secretion, since in the AQP5-null mice saliva secretion was remarkably reduced, but unimpaired in AQP1 or AQP4-null mice173. AQP5 is also expressed in the apical membrane of tear-secreting cells of the lacrimal gland and was implicated in the pathophysiology of Sjögren's syndrome (an autoimmune disease characterized by dry eyes and a dry mouth). two groups of investigators reported a decrease in AQP5 levels in the salivary gland [Steinfeld et al., 2001, cited in171] and lacrimal gland secretory cells [Tsubota et al., 2001, cited in171]. However, a third group found no difference in the AQP5 distribution in salivary gland of a larger cohort of patients with Sjögren's syndrome compared with control subjects [Beroukas et al., 2001, cited in171].

AQP4 is expressed in the stomach parietal cells, where stomach acid is produced; however AQP4 does not appear to facilitate gastric acid production. AQP4 is also expressed in the colon, probably playing a minor role in the dehydration of feces173.

AQP3, AQP5, AQP8, AQP9, and AQP10 have also been found in the epithelium along the GI tract; out of these, AQP10 is expressed exclusively in the small intestine, in the absorptive epithelial cells171.

AQP1 was reported to be expressed at several sites in the proximal GI tract, possibly playing a role in dietary fat processing, including cholangiocytes in the liver (bile production), pancreatic microvascular endothelium (pancreatic fluid production), intestinal lacteals (chylomicron absorption) and gallbladder (bile storage)173. Recently, Jin et al.184 described a broad distribution of AQP1 in epithelium and endothelium of porcine digestive organs, and suggested an important role in fluid secretion/absorbtion, as well as in digestive function and pathophysiology of the GI system. However, this work was critically evaluated by Mobasheri185.

Other aspects regarding WCPSs in the GI tract and organs are discussed by Koyama et al., 1999; Ma and Verkman, 1999; Matsuzaki et al., 2004, [cited in171]; see also ref.186, for a review on WCPSs in the exocrine pancreas and refs.187, 188 for reviews on WCPSs in control of volume in secretory vesicles.

The respiratory apparatus (airways, lung, pleura) has potentially important sites of fluid movement in airway hydration, reabsorption of alveolar fluid in the neonatal period, formation/resolution of pulmonary edema, resulting from heart failure or lung injury, pleural fluid secretion/reabsorbtion and accumulation in pathological conditions189. In the upper respiratory tract, the epithelium of the nasopharynx and trachea has AQP3 in basal cells, AQP4 in the basolateral

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membrane of ciliated columnar cells, and secretory cells in the submucosal glands, and AQP5 in the apical membrane of these secretory cells. In the epithelium of distal airways there is only AQP4, whereas in the alveolar epithelium AQP5 is present in the apical membrane of type-I pneumocytes. AQP1 is present in pleura and microvessels (capillaries and venules) throughout all components of the respiratory apparatus [reviewed in171, 188]. This expression pattern of WCPSs provides indirect evidence for their involvement in fluid handling by lung and airways. Several other lines of evidence appear to support physiological roles of AQPs in lung and airways. The expression of lung AQPs is developmentally regulated with distinct patterns for each AQP. AQP1 is detectable just before birth in rodents, increasing several-fold perinatally and into adulthood, while rabbit lung Water permeability in the perinatal period parallelled increasing AQP1 expression. AQP4 strongly increases just after birth and is upregulated by β-agonists and glucocorticoids; in contrast, little AQP5 is expressed at birth and gradually increases until adulthood [see citations in189]. Many other observations of increased or decreased expression of lung AQPs induced by growth factors, inflammatory mediators, viral infection, osmotic stress, lipopolysaccaride or nickel-induced lung injury, cAMP, tumor necrosis factor-α have been published [reviewed in171, 189]. However, studies on aquaporin null-mice suggested that although AQPs provide a major route for osmotically-driven Water transport among the airspace, interstitial and capillary compartments, AQP1, AQP3, and AQP4 are not required for physiologically important lung functions189. A notable exception is AQP5, wich appeared to be important in fluid secretion by submucosal glands in the upper airways. Consequently, modulation of AQP5 expression or function could provide a novel therapy to change the volume and viscosity of fluid secretions in cystic fibrosis and infections of allergic rhinitis189.

WCPSs in the central nervous system (CNS) appear to be of great physiological and pathological importance, considering the rigid physical constraint that is imposed to the brain by the bony cranium and that ∼80% of the brain is Water190.

In rodent brain cells seven WCPSs have been described: AQP1, AQP3, AQP4, AQP5, AQP8, AQP9, and AQP11, however only three have been clearly identified in the apical (but not the basolateral) membrane of brain cells in vivo: AQP1, AQP4, and AQP9191.

AQP1 is expressed in epithelial cells of the choroid plexus (CP)192 and is probably involved in the cerebrospinal fluid (CSF) formation193. This idea is supported by the increased CSF production in CP tumours in parallel with increased expression of AQP1. As hydrocephalus is associated with CSF flow abnomalities, inhibitors of AQP1 might be useful in treating this disease190. Interestingly, AQP1 is not found in the normal brain capillary endothelium, although is highly expressed in peripheral endothelial cells. AQP1 is present in capillaries and astrocytes of astrocytomas and metastatic carcinomas194. AQP1 was also found in small-diameter sensory neurons in dorsal root, trigeminal and nodose ganglia and colocalized with markers of nociceptors, notably substance P. Impaired pain sensation in AQP1-null mice has been reported by Oshio et al. 2006, [cited in195]. It was therefore suggested that AQP1 may be involved in the pathophysiology of migraine195.

AQP4 is expressed strongly throughout the brain and spinal cord, especially in astroglial cells lining ependyma and pial surfaces in contact with the CSF and the blood-brain barrier, in glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of the hypothalamus, and Purkinje cells of cerebellum190. This pattern of distribution is in agrement with the major role of AQP4 to control Water movements into and out of the brain.

A role of AQP4 in the generation of brain edema in response to two established neurological insults (acute Water intoxication and ischemic stroke) has been proposed196. AQP4-null mice appeared to be protected from brain edema in both models, with improved clinical outcome and reduced brain swelling [reviewed in173]. On the other hand, several studies have shown that the expression of AQP4 is reduced or increased after ischemia-induced brain edema depending on the brain region and the time after the onset of the ischemic insult [Frydenlund et al., 2006; Ribeiro et al., 2006, cited in197]. Reduced brain swelling after cerebral ischemia and Water intoxication has also been reported in α-syntrophin-null mice, which have reduced AQP4 expression in astrocyte foot processes [Amiry-Moghaddam et al, 2003; 2004, cited in190]. In contrast to its beneficial role in cytotoxic edema, AQP4 deficiency produces more brain swelling in mouse models of vasogenic edema, including brain tumor, infusion of normal saline into brain extracellular space and focal cortical freze injury [Papadopoulous et al., 2004, cited in190]. Based on the earlier discussion it was suggested that AQP4 inhibitors may reduce cytotoxic brain swelling in humans, whereas AQP4 activators or upregulators may reduce vasogenic edema and hydrocephalus190.

A second role of AQP4, in astrocyte migration, has been suggested based on impaired migration of cultured AQP4-null astrocytes compared to wild-type astrocytes. On the other hand AQP4 overexpression is a feature of astrocytomas, facilitates cancer spread and AQP inhibitors may slow tumor growth190.

A third role of AQP4 in brain was suggested to be the control of neuronal activity. It has been proposed that altered K+ kinetics in brain ECS account for the altered neuronal activity in AQP4 deficiency [reviewed in190, 191]. In vivo, AQP4-null mice have delayed K+ clearance from brain ECS after local electrical stimulation [Binder et al., 2006, cited in190] and during cortical excitation by spreading depression [Padmawar et al., 2005, cited in190]. Hippocampal slices from α-syntrophin-null mice also have slowed K+ kinetics after evoked neuronal activity, and the altered K+ kinetics in AQP4 deficiency has been explained by a functional association between AQP4-facilitated Water movement and K+ movement through the Kir4.1 [Amiry-Moghaddam et al., 2003, cited in190]. However, no functionally significant interactions between AQP4 and Kir4.1 were found [reviewed in190].

ECS expansion has recently been proposed as an alternative mechanism to account for higher seizure threshold and prolonged seizure duration in AQP4 deficiency198. An increased ECS volume increases the buffering capacity for K+

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released into the ECS during neuronal excitation, preventing large changes in ECS [K+]. It is unclear, however, why the ECS volume is increased in AQP4-null mice190. All these findings confirm our earlier conclusion of a membrane defect affecting Water permeability in epilepsy [reviewed in199]. The seizure phenotype data in AQP4-deficient mice raise the possibility that AQP4 modulation may also be effective in epilepsy therapy197.

Another physiological role has been suggested for AQP4 in cell ADHesion between astrocytes and endothelial cells or muscle cells in the perivascular compartment. The presence of AQP4 of the endfoot membrane is dependent upon the presence of proteins in the basal lamina such as agrin, α-dystroglycan and laminin [Guadagno and Moukhes, 2004, cited in191], suggesting an involvement of AQP4 in the ability of astrocytes to maintain the integrity of blood-brain barrier.

Recently, it has been found that autoimmune reactions with autoantibodies against AQP4 appear to produce neuromyelitis optica (Devic's disease) and the presence of these autoantibodies is a criterion for differential diagnosis with multiple sclerosis200.

AQP9 has been observed in three cell types: endothelial cells of sub-pial vessels, glial cells (in particular tanycytes and astrocytes), and neurons [reviewed in191]. AQP9 expression was found predominantly in one subtype of neurons, the catecholaminergic neurons [Badaut et al., 2004, cited in191]. These neurons are involved in energy balance [Grill and Kaplan, 2002, cited in191]. Consequently, a role for AQP9 in brain energy metabolism have recently been proposed. AQP9 facilitate the transport of lactate across the astrocyte membrane and lactate diffuses to neurons for energy consumption [Pellerin et al., 2007, cited in190]. AQP9 permeability to Water, glycerol and lactate may be important in brain ischemia. Lactic acidosis during ischemia may increase the permeability of AQP9 and enable uptake of excess lactate by astrocytes.

Water homeostasis in the eye, involving protection of the epithelium, regulation of intraocular fluid levels and pressure, maintaining the transparency of the pathway for light (cornea and lens), retinal signal transduction, are crucial processes for the normal functioning of the eye201.

The cornea consists of a stromal layer, covered at its external surface by an epithelium, in contact with tear fluid, and at its inner surface by an endothelium, in contact with aqueous humor (AH) (the fluid in anterior eye chamber). two AQPs are present in mammalian cornea: AQP1 in endothelial cells and AQP5 in epithelial cells. AQP1 is involved in extrusion of fluid from the corneal stroma across the corneal endothelium and AQP5 in a similar process across the corneal epithelium. As a result both transparency and normal thickness (determined by hydration) of cornea depend upon AQP1 and AQP5. In AQP1-null mice the thickness of cornea is decreased, while in AQP5-null mice the thickness is increased202. In addition, AQP5 probably contributes to the generation of surface liquid that helps to protect cornea from mechanical injury171.

AQP3 found in conjunctiva (the epithelium that covers the outer margins of the eye) might help to lubricate the surface of the eye171. Recently, Candia and cowokers203 reported the presence of AQP5 in mammalian conjunctiva suggesting that AQP5 could be a potential target for pharmacological upregulation that may enhance fluid secretion in individuals with dry-eye syndromes.

AQP1 and AQP4 are present in the nonpigmented epithelium of the anterior ciliary body, a structure that contributes to the movement of AH; in addition, AQP1 is present in the trabecular meshwok and canals of Schlemm, structures that resorb AH out of the anterior chamber [Hamann et al., 1998, cited in171].

AQP1 is also present in the lens epithelium, whereas AQP0 constitutes 50% of the total membrane protein in the fibre cells of the lens. Recently, accelerated cataract formation was found in AQP1-null mice202. AQP0 was discovered in 1984 (MIP26) and only in 1995 it was identified as a WCPS [Mulders et al., 1995, cited in201]; although it has a low permeability to Water it contributes to the maintenance of lens transparency; mutations in AQP0 are associated with hereditary cataracts in humans [reviewed in171]; AQP0 also has a structural role as a cell–cell ADHesion molecule204.

AQP4 is expressed in Müller cells in retina, colocalized with the K+ channel Kir4.1. Müller cells are supporting cells associated with bipolar cells, similarly to astrocytes in the CNS, associated with neurons [see refs. in201]. It was thought that AQP4 modulates Kir4.1 K+ channel function. However, recently Ruiz-Ederra and Verkman205 provided evidence against functional interaction between AQP4 and Kir4.1K+channel in retinal Müller cells.

There are two WCPSs in adipose tissue: AQP1 and AQP7. Although AQP1 is abundant in adipocytes its functional significance is not yet precisely known. Many studies have been performed on AQP7, reviewed in51, 206. Adipocytes hydrolyze or synthesize triglycerides (TG) in response to the whole body energy balance. The rapid increase in glycerol production during lipolysis results in rapid increase in intracellular osmotic pressure, which could damage the cell 206 ; hence, a possible function of AQP7 as a glycerol channel in adipocytes appears of great importance. However, there is uncertainty as to the precise cellular localization of AQP7 in adipose tissue. Most researchers assume that AQP7 is localized in the adipocyte membrane51, while Skowronski et al.207 reported that it is actually expressed in the capillary endothelia of adipose tissue (and cardiac and striated muscle). More studies are necessary to solve this controversy and clearify how does glycerol exit the adipocyte and what is the function of AQP7 in the vessels51.

It seems that obesity is associated with a dysregulation of AQP7. Several AQP7-KO model mice were generated by different gene targeting strategies and, interestingly, show different phenotypes. The main difference between the different KOs is in their susceptibility to developing obesity [discussed in51]. The recent discovery of AQP7 in the pancreatic β-cells generated a novel hypothesis that the primary reason for the metabolic disturbance of the AQP7-KO mice may be the malfunctioning of β-cells rather than adipocytes208. A potential role of AQP7 in human obesity and, eventually through body weight changes in the development of type II diabetes is suggested by reports of decreased AQP7 expression in

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obese subjects209. More studies are necessary to clarify the role of AQP7 from adipose tissue and/or pancreatic β-cells in the development of obesity and the various metabolic disturbances observed in AQP7-KO mice.

In the skin AQP1 has been detected in the rat dermal capillaries [Agre et al., 2003 cited in210] and human neonatal dermis; [Marchini et al., 2003, cited in190]. AQP1 and AQP3 have also been found in epidermis and dermis in erythema toxicum neonatorum [Marchini et al., 2003, cited in210]. Special attention has been paid to the roles of AQP3 in the epidermis, where it is present in the plasma membrane of the basal cells and the adjacent intermediate cells170 and completely disappears in the outermost epidermal layer, the keratinized layer, also called stratum corneum (SC). AQP3 is also present in the associated structures of the epidermis such as hair follicles and sebaceous glands (including the Meibomian gland of the eyelid)170. SC is composed of keratinocytes embedded in a complex mixture of nonpolar lipids and serves as a barrier against the evaporation of Water from the skin. Matsuzaki et al. [cited in170] proposed that AQP3 in epidermal cells serves as machinery to supply Water to the Water-deprived epidermal cells from the underlying dermis where capillaires provide enough Water from the blood, followed by transfer of Water among cells (including highly differentiated cells in the upper layer of the epidermis) via gap junction intercellular channels. The crucial role of AQP3 in the hydration of the epidermis is supported by findings in AQP3-null mice: impaired skin hydration, elasticity and barrier function [reviewed in210].

Cao et al.211 found that ultraviolet (UV) radiation induces downregulation of AQP3 in keratinocytes, which result in reduced Water permeability, decreased cell migration and delayed wound healing; these effects are efficiently counteracted by all-trans retinoic acid. UV radiation decrease the Water content and Water-holding capacity of SC; the deprived Water content caused by UV radiation further damages the function of SC, leading to deleterious effects such as wrinkle formation and delayed wound healing [Weiss et al., 1988, cited in211].

On the other hand Hara-Chikuma and Verkman210 considered that glycerol rather than Water transporting function of AQP3 is important in skin physiology. The dry, relatively inelastic skin in AQP3-null mice was correlated with the humectant properties of glycerol and the impaired SC repair to impaired glycerol biosynthetic function. The authors suggested that the key role of AQP3 in epidermal physiology might be exploited in the development of improved cosmetics and therapies for skin diseases associated with altered skin Water content. In addition, it was found212 that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. The authors suggested that AQP3-facilitated transport in epidermal cell represents a novel mechanism of cell proliferation and tumorigenesis implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy. This suggestion is also based on finding of greatly increased AQP3 expression in human skin squamous cell carcinoma212.

In the last decade many other studies regarding the possible implications of AQPs in cancer have been performed. The results are, however, conflicting. Some authors reported a decrease or loss of AQP1 in renal cell carcinoma, suggesting even a posible role of AQP1 as a prognostic or tumor grading marker; in contrast, other authors reported overexpression of AQP1 in lung and bronchoalveolar carcinoma [see citations in213]. In brain tumors (glioblastomas) AQP1 expression was found to increase with the grade of malignancy and the deletion of AQP1 in genetically modified mice reduces angiogenesis, a process essential for growth and spread of tumors194. In subsequent in vitro studies Hu and Verkman214 found that AQP1 expression in melanoma cell increased their migration and metastatic potential, suggesting a novel function for AQP expression in high-grade tumor and pointing to AQPs as posible targets in tumor therapy. Other authors reported heterogenous expression of AQP1 and other AQPs in a variety of tumors [see citations in215].

PERSPECTIVES Since the discovery in 1985 of the first WCPS, hundreds of such proteins have been identified in organisms from all kingdoms of life. It is clear that WCPSs (and MIPs) play major roles in a broad array of basic physiological processes in all cells and organisms in which such proteins are expressed. However, future research should clearify the physiological roles of MIPs in organisms and cells where the role is not yet obvious; such is the case of some MIPs in microorganisms, of SIPs in plants, of other subcellular AQPs in animals and humans. Future work is needed to discover new members of the MIP superfamily in diverse organisms and to assign them a physiological role. An important direction of research is represented by studies leading to new methods for diagnosis and therapy of diseases in veterinary and human medicine based on progress in understanding the precise cellular localization and the regulation of expression of WCPS genes in various cells. Modulation of WCPS expression or function by inhibitors or activators of channel (pore) permeability could provide novel drugs or therapies in various diseases, from tropical parasitic disorders to genetic disease (e.g. cystic fibrosis), from obesity and metabolic diseases to cancer.

Acknowledgements The author acknowledges the collaboration over the past 30 years of all coauthors in their publications listed in references. The financial support from Romania (“Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca, Ministry of Education and Research, National Council for Higher Education Scientific Research, Academy of Medical Sciences, National Council for Science and Technology), UK (The Wellcome Trust, The British Council), USA (National Science Fondation), and Australia (Taronga Zoo, Sydney) for the work reviewed in this article is gratefully acknowledged. He thanks several distinguished colleagues for sending him publications used in the elaboration of this review and/or for critical reading of the manuscript: Eric Beitz (Tübingen, Germany), Cong Cao (Nanjing, China), Robert A. Fenton (Arhus, Denmark), Tamir Gonen (Boston, USA), Stefan Hohmann, Karin Linkvist and Martin Markström (Gothenburg, Sweden), Vincent J. Huber (Niigata, Japan), Xuejun Li (Beijing, China), Nancy Kaufman (Pitsburgh, USA), Christophe Maurel (Montpellier, France), Richard Naftalin (London, UK), Shigeyasu Tanaka and Masakazu Suzuki (Shizuoka, Japan), Aurel Popa-Wagner (Greifswald, Germany), An Tanghe (Leuven, Belgium), Chang

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