Insights into structural mechanisms of gating induced regulation of aquaporins.

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JPBM898_proof ■ 5 February 2014 ■ 1/11

Progress in Biophysics and Molecular Biology xxx (2014) 1e11

Contents lists available at ScienceDirect

Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio

Review

Insights into structural mechanisms of gating induced regulation of aquaporins Q2

Ruchi Sachdeva a, *, Balvinder Singh b a b

MCM DAV College, Sector 36A, Chandigarh, India Bioinformatics Center, Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160036, India

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation. Ó 2014 Published by Elsevier Ltd.

Keywords: Aquaporin Regulation Gating Phosphorylation Molecular dynamics simulation Water permeability

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gating of plant aquaporin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aquaporin-4 gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeast aquaporin gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AQP2 gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AQPZ gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gating of aquaporin-0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Aquaporins are ubiquitous family of membrane proteins that facilitate the rapid transport of water across cell membranes and thus play a critical role in maintaining water homeostasis in all living cells. In cell membranes, aquaporins exist as homotetramers

* Corresponding author. Tel.: þ91 9876481718. E-mail address: [email protected] (R. Sachdeva).

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with each monomer forming an independent water channel. Aquaporin family members mediate the bidirectional water flow driven by an osmotic gradient. They enable highly efficient water permeation with flow rates in order of 109 s1 (Zeidel et al., 1992). In addition to water, some aquaporin family members, called aquaglyceroporins transport certain small, neutral solutes such as glycerol (Heller et al., 1980; Borgnia and Agre, 2001), ammonia (Saparov et al., 2007), urea (Borgnia et al., 1999), arsenite (Liu et al., 2002). Aquaporins have a remarkable property of effective water conductance while blocking the flow of protons and thus maintain

0079-6107/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pbiomolbio.2014.01.002

Please cite this article in press as: Sachdeva, R., Singh, B., Insights into structural mechanisms of gating induced regulation of aquaporins, Progress in Biophysics and Molecular Biology (2014), http://dx.doi.org/10.1016/j.pbiomolbio.2014.01.002

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R. Sachdeva, B. Singh / Progress in Biophysics and Molecular Biology xxx (2014) 1e11

the electrochemical gradient across cell membranes (de Groot et al., 2003). In eukaryotic organisms, aquaporins perform a wide variety of physiological functions, such as concentrating urine in kidneys (Chen et al., 2005), maintaining lens transparency in eyes (Verkman, 2003) maintaining water homoeostasis in brain (AmiryMoghaddam and Ottersen, 2003), cell migration during tumor growth (Saadoun et al., 2005), facilitating a rapid response to shock in yeast (Tamas et al., 1999), regulation of cell osmolarity in plants (Maurel et al., 2002), driving the opening and closing of flower petals (Azad et al., 2008). Physiological significance of aquaporins is further highlighted by the fact that a number of diseases including brain edema (Manley et al., 2000), tumor (Vacca et al., 2001), obesity (Kuriyama et al., 2002) manifest abnormal functioning of these water channels. A lot of structural information has been deduced from primary sequence of aquaporins. Sequence analysis of AQP1 has revealed two tandem repeats each formed from three transmembrane domains with two highly conserved loops (B and E) containing the signature motif, asparagines-proline-alanine (NPA). The repeats have been predicted to be oriented 180 with respect to each other. Based on biochemical and site-directed mutagenesis studies, an ‘hourglass model’ has been proposed according to which loops B and E fold back into the bilayer from the opposite sides of the membrane thereby forming the aqueous pore (Jung et al., 1994a). Aquaporin is emerging to be the richest family of membrane channels with regard to the abundance of high resolution structural data. Three dimensional crystal structures of several members of the family have been solved. The structures of 2 bacterial AQP: GlpF, glycerol channel (Fu et al., 2000) and AqpZ (Savage et al., 2003); 7 mammalian AQP: bovine and sheep AQP0 (Gonen et al., 2004; Harries et al., 2004), human AQP1 (Murata et al., 2000), bovine AQP1 (Sui et al., 2001), rat AQP4 (Hiroaki et al., 2006), human AQP4 (Ho et al., 2009), human AQP5 (Horsefield et al., 2008); SoPIP2;1 (spinach) aquaporin (Tornroth-Horsefield et al., 2006); archaeal AQP (Lee et al., 2005), PfAQP from malarial parasite Plasmodium falciparum (Newby et al., 2008) and yeast AQP (Fischer et al., 2009) are available in PDB. Elucidation of three dimensional structures of aquaporin family members has confirmed the hourglass fold that

was previously suggested by sequence analysis. As shown in Fig. 1a, the hourglass fold consists of six transmembrane a-helices surrounding a single, narrow aqueous pore (Sui et al., 2001). Loops B and E form half transmembrane helices (HB and HE) and fold into the channel from opposite sides of the membrane, effectively creating a seventh broken transmembrane helix (Fig. 1a). The Nterminal ends of these half helices contain the aquaporin Asn-ProAla (NPA) signature motifs that meet at the center of the pore (Fig. 1b). Aquaporins contain a few highly conserved residues in and around the two functional loops B and E that are closely positioned in the interior of the membrane. In case of bAQP1, these are residues Leu-77, His-76, Ala-75 and Gly-74 present on loop B and residues Gly-190, Cys-191, Gly-192 and Ile-193 located on loop E. Loops B and E are held together by van der Waals interactions between the prolines in the two NPA motifs. The positions of loops B and E are stabilized through ion pairs and hydrogen bonds with neighboring transmembrane helices. Molecular dynamics simulation of aquaporins has provided useful insights into mechanism of water permeation. Simulations have revealed that water molecules move in a single file configuration through the channel. The two half helices HB and HE generate electrostatic fields directed toward the center of the channel, thereby creating an electrostatic barrier which results in a complementary alignment of the dipole moments of water molecules as they move past the NPA motifs (Tajkhorshid et al., 2002). Starting from the NPA motifs, water molecules are oriented in opposite direction in the two halves of the channel. Above and below the NPA motifs, hydrogen atoms of water molecules point towards extracellular mouth and cytoplasmic mouth of the channel respectively. This results in the bipolar orientation of water molecules inside the aquaporin channel as shown in Fig. 2. The electrostatic barrier of approximately 25e30 KJ mol1 is suggested to be the predominant cause of proton exclusion (de Groot et al., 2003; de Groot and Grubmuller, 2005). Another conserved structural feature of aquaporin family is the aromatic/arginine (ar/R) constriction site located at the extracellular side of the channel (Fig. 1b). The ar/R constriction site which consists of an arginine and other three amino acids (one of which is usually aromatic) such as

Fig. 1. Structural features of aquaporin family members. (a) The hourglass fold. Transmembrane helices are denoted as H1eH6, loops are denoted as AeE and the two pore helices formed by loops B and E are denoted as HB and HE respectively. (b) Details of NPA motifs and ar/R site. Water molecules inside the channel are shown as red spheres. The structure of bovine AQP1 (PDB ID 1J4N) is used to show the common fold.


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With such an interesting structural biology of aquaporins, one prompts to further understand aquaporins by asking how water permeation through aquaporins is regulated. The transport of water across aquaporin channel is regulated either by gating, i.e. the rate of water flow through the channel is controlled or by trafficking which includes targeting of aquaporins to different membranes. Numerous biochemical studies have revealed the key role of phosphorylation of serine or threonine residue in gating as well as trafficking of aquaporins (Johansson et al., 1998; King et al., 2004; Hoffert et al., 2008; Prak et al., 2008; Woo et al., 2008). Other common signals through which aquaporins are regulated include changes in pH (Nemeth-Cahalan and Hall, 2000), changes in divalent cation concentrations (Gunnarson et al., 2005) and osmolality (Conner et al., 2010). High resolution structures of aquaporins and molecular dynamics simulations have uncovered the structural mechanism of aquaporin gating. In this review, regulatory mechanisms of gating in several aquaporins are discussed.

2. Gating of plant aquaporin Fig. 2. Bipolar orientation of water molecules inside the bovine AQP1 channel. Asparagines of the two NPA motifs are shown as CPK model.

His, Phe and Cys in case of bAQP1, acts as a selectivity filter. The ar/R constriction site of bAQP1 is approximately 2.8 A in diameter, comprising of amino acid residues Arg-197, His-182, Phe-58 and Cys-191 (Sui et al., 2001). In comparison to AQP1, only arginine is conserved in ar/R site of aquaglyceroporin such as Escherichia coli GlpF whereas Phe-58, His-182 and Cys-191 of bAQP1 are replaced by Trp-48, Gly-191 and Phe-200 in GlpF, respectively (Fu et al., 2000). The His / Gly substitution provides room needed to accommodate additional Phe / Trp and Cys / Phe substitutions, resulting in increase in both size and hydrophobicity of ar/R site of GlpF. The resulting constriction site of GlpF is larger in diameter of w3.8 A and amphipathic, thereby allowing bigger solute i.e. glycerol to pass through the channel. Moreover, it is believed that not only diameter but polarity of ar/R constriction site is also crucial for determining the substrate specificity of aquaporins (Savage et al., 2003; Wang et al., 2005).

In plants, cellulose cell wall functions to maintain a large osmotic gradient between the cell interior and exterior that is crucial for plant physiology and gives plant cells their structure and rigidity. Plants have developed complex systems of regulating water transport in order to cope with rapid changes in water availability. In plants, there are 35 aquaporin family members which are further divided into four subfamilies based on phylogenetic analysis (Johanson et al., 2001). All plant aquaporins that lie within the plasma membrane, i.e. PIPs are gated where the channel closure is triggered either by the dephosphorylation of two highly conserved serine residues (Johansson et al., 1998) or by a drop in the pH (Tournaire-Roux et al., 2003). There is 98% reduction in water transport activity of PIPs by a drop to acidic cytoplasmic pH or addition of Caþ2 from the cytoplasm (Alleva et al., 2006). In spinach plasma membrane aquaporin SoPIP2;1, pH gating is governed by the protonation state of a highly conserved His-193 in loop D (Johansson et al., 1998). Opening of SoPIP2;1 channel is achieved by phosphorylation of Ser-115 in cytoplasmic loop B and Ser-274 in the C-terminal region, both of which lie in consensus phosphorylation

Fig. 3. Top view from the cytoplasmic side of SoPIP2;1 channel. (a) Closed conformation and (b) Open conformation of SoPIP2;1 structure. Loop D (highlighted in red) exhibits different conformation in open and closed states of this water channel.


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sites (Johansson et al., 1998). High-resolution X-ray structures of SoPIP2;1 in closed and open conformations and molecular dynamics simulations have provided useful insights into the structural mechanism of phosphorylation induced gating (TornrothHorsefield et al., 2006). The closed conformation of SoPIP2;1 has revealed that loop D caps channel entrance from the cytoplasm as shown in Fig. 3a (Tornroth-Horsefield et al., 2006). Due to this, the blocking residue, Leu-197 gets inserted into a cavity near the entrance of channel and in combination with other residues, it creates a hydrophobic gate that blocks the channel. In open structure of SoPIP2;1, the loop D is entirely displaced by a half-helix extension of helix 1 into the cytoplasm, thereby removing Leu197 from the cytoplasmic entrance and thus, opening the channel (Fig. 3b). The N terminus of SoPIP2;1 contains a divalent cation binding site occupied by Cdþ2 in the crystal structure, but presumably Caþ2 in vivo. A network of ionic and hydrogen bond interactions extending from an N-terminal divalent cation binding site anchors loop D to the N-terminus and is critical in defining the closed conformation of SoPIP2;1. Hydroxyl group of Ser-115 side chain forms a hydrogen bond to Cdþ2 ligand Glu-31 as revealed in X-ray structure of closed conformation of the aquaporin (TornrothHorsefield et al., 2006). Phosphorylation of Ser-115 has been thus speculated to disrupt this network of hydrogen bonds and thus would allow loop D to move from its closed to open conformation. Further, molecular dynamics simulations of SoPIP2;1 containing phosphorylated Ser-115 has revealed partial opening of the hydrophobic gate mediated by displacement of loop D within 15 ns (Tornroth-Horsefield et al., 2006). This mechanism can also explain the phenomenon of SoPIP2;1 channel closure triggered by the protonation of His-193 (Alleva et al., 2006) which is located on the opposite side of the Cdþ2 binding site to Ser-115. Upon protonation, His-193 would form a strong ionic interaction with Asp-28 (another

Cdþ2 ligating residue) and anchor loop D to the N terminus, thereby keeping the hydrophobic gate closed. The phenomenon of channel opening induced by phosphorylation of Ser-274 located near the Cterminus can also be explained within this framework (Johansson et al., 1998). The side chain hydroxyl group of Se-274 forms hydrogen bond interactions with the backbone nitrogen atoms of Pro-199 and Leu-200 from an adjacent monomer in the closed structure of SoPIP2;1. Phosphorylation of Ser-274 would disrupt these hydrogen bonds thereby releasing Ser-274 and causing a disorder of the C-terminal region that would prevent a steric clash between Ser-274 and Leu-197 of neighboring monomer. Helix 5 may thus extend an extra half turn into the cytoplasm as seen in the open conformation of SoPIP2;1. Unexpectedly, X-ray structures of S115E and S274E single SoPIP2;1 mutants and S115E:S274E double mutants have been shown to have a closed conformation (Nyblom et al., 2009). This implies that neither substitution fully mimics the phosphorylated state. The mutant structures of SoPIP2;1 have been observed to be similar to wild type closed conformation of SoPIP2;1 (Fig. 4). Any significant conformational change has not been observed upon substitution of Ser-274 except that it disorders the C-terminus. Structural differences have been observed between wild type and mutant SoPIP2;1 structures near Ser-115. Mutation of Ser-115 has been shown to induce a half-turn extension of transmembrane helix 1, which draws the Ca atom of Glu-31 away from its wild type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Despite the disruption of divalent cation binding site in S115E mutant, loop D was found to adopt the same conformation as in the wild type closed structure and remained anchored to loop B through hydrogen bond interactions. However, details of these interactions have been found to differ when compared with the wild type. Several of the water mediated

Fig. 4. Structural comparisons of wild type and mutant SoPIP2;1. (a) Structural superimposition of open conformation of wild type SoPIP2;1 (pink; PDB ID 2B5F) and double mutant of SoPIP2;1 (cyan; PDB ID 3CN5). (b) Structural superimposition of closed conformation of wild type SoPIP2;1 (yellow; PDB ID 1Z98) and double mutant of SoPIP2;1 (cyan; PDB ID 3CN5).


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hydrogen bond interactions between loop D, loop B and the Nterminus were disrupted in the mutant structures, suggesting that the effective local structural perturbations associated with the phosphorylation of Ser-115 are larger than those observed for the S115E and S115E:S274E mutants. The possible reason for the failure to fully mimic the phosphorylated protein by replacing the phosphorylatable residue by glutamate has been attributed to the difference in total charge on the respective side chains, with the phosphorylated side chain predominantly carrying a 2 charge at physiological pH compared to the singly negatively charged carboxylate group on the glutamate (Nyblom et al., 2009). Inspection of hydrogen bonds within loop D has suggested an important role of phosphorylation of Ser-188 in opening the channel. This finding has been supported by increased water transport activity of S188E mutant. Moreover, MD simulations have revealed interactions between phosphorylated Ser-188 of loop D and Lys-270 of C-terminus leading to a conformational change of loop D and channel opening. This study has described for the first time, an active role of C-terminus in gating of SoPIP2;1 (Nyblom et al., 2009). There are additional reports suggesting pH regulation of Arabidopsis thaliana and Vitis vinifera PIP aquaporins heterologously expressed in Xenopus oocytes (Tournaire-Roux et al., 2003; Shelden et al., 2009). Such plants respond to conditions that lead to a decrease in cytosolic pH by reducing the water permeability of cell membrane. In another report, pH regulation of Vitis vinifera cv. Touriga nacional TIP2;1 (VvTnTIP2;1) water channels cloned in yeast has been studied (Leitao et al., 2012). Water permeability of VvTnTIP2;1 has decreased by approximately 50% when there was a drop in cytosolic pH from 6.8 to 4.8. VvTnTIP2;1 sequence possesses a histidine residue (His-131) in loop D. This histidine is commonly found in plant PIPs, such as His-193 in SoPIP2;1, His-194 in Arabidopsis thaliana PIP2;1. Role of histidine in pH regulation of plant aquaporins has already been elucidated (Tornroth-Horsefield et al., 2006). Mutation of His-131 to aspartic acid or alanine has resulted in loss of pH dependent decrease in water permeability of VvTnTIP2;1, suggesting a possible gating mechanism (Leitao et al., 2012). Further structural studies on VvTnTIP2;1 are required to fully understand the structural changes caused upon change in pH. 3. Aquaporin-4 gating Aquaporin-4 is a predominant water channel found in the mammalian brain (Jung et al., 1994b). Activation of protein kinase C has been shown to decrease the water permeability of rat aquaporin-4 (rAQP4) expressed in the basolateral membrane of kidney epithelial cells (Zelenina et al., 2002). Phosphorylation of Ser-180 has been shown to be involved in the regulation of rAQP4 by protein kinase C activation. This regulation of rAQP4 has been suggested to occur via gating since there was no change in the subcellular distribution of aquaporin upon activation of protein kinase C. Moreover, when serine was mutated into alanine, the protein kinase C mediated decrease in water permeability was abolished, indicating direct phosphorylation of rAQP4 by protein kinase C that results in an allosteric change in the aquaporin leading to decreased water permeability (Zelenina et al., 2002). Since Ser-180 is present near the cytoplasmic mouth of channel, phosphorylation of this residue is likely to increase chances of its interactions with C-terminal region of rAQP4. Phosphorylated Ser180 has been speculated to interact with positively charged residues present in the C-terminus (Lys-259, Arg-260, Arg-261) of rAQP4 (Hiroaki et al., 2006). Blocking of cytoplasmic mouth of the channel by this interaction may explain gating of rAQP4 due to phosphorylation (Hiroaki et al., 2006). In another study, activation of vasopressin receptor has been shown to induce a dramatic decrease in water permeability of

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rAQP4 expressing Xenopus oocytes (Moeller et al., 2009a). However, this effect has been attributed to protein kinase C dependent internalization of the water channel. Substitution of Ser-180 of rAQP4 to alanine has been shown to decrease the protein kinase C dependent reduction in water permeability and rAQP4internalization. This suggests a role of Ser-180 in the regulation of rAQP4 by protein kinase C dependent internalization of aquaporin rather than by gating and thus contradicts the previous findings on regulation of this water channel (Zelenina et al., 2002; Moeller et al., 2009a). In order to mimic the phosphorylated Ser-180, the structure of AQP4S180D mutant has been solved to 2.8 A resolution by electron diffraction (Mitsuma et al., 2010). Interestingly, the mutant structure does not show any significant differences from the wild type rAQP4, suggesting that S180D mutation does not induce large conformational changes in rAQP4 structure. Analysis of pore profiles has shown that the cytoplasmic side of the channel is wider in the S180D mutant than in wild type rAQP4. Water conductance measurements, both in vitro and in vivo, have shown the same water permeability for wild type and AQP4S180D mutant that further confirms the open conformation of mutant rAQP4 channel (Mitsuma et al., 2010). Further insights into the mechanism of rAQP4 regulation have been provided by MD simulations of both unphosphorylated rAQP4 and rAQP4 phosphorylated at Ser-180 (Sachdeva and Singh, 2013). Rat AQP4 has an approximately 70 residues long C-terminus which is much larger than that of other aquaporins. Since the role of residues of C-terminus in rAQP4 regulation is speculated, the Cterminal region starting from 255 to 323 residues has been modeled in the study. MD simulations have revealed interactions between phosphorylated Ser-180 and positively charged residues located on C-terminal region and cytoplasmic loops (Fig. 5a). Each of the monomers of rAQP4 exhibit different set of interactions between phosphorylated Ser-180 and positively charged residues. These interactions have been found to take place on the same side of the cytoplasmic mouth and stabilize the already open conformation of loop D, thus keeping it away from the channel entrance (Fig. 5b). Movement of C-terminal region towards cytoplasmic mouth has not resulted in closure of the channel’s entrance (Fig. 6). A continuous flow of water molecules from aqueous phase till cytoplasmic mouth has been observed in unphosphorylated as well as in phosphorylated rAQP4. Osmotic permeabilities of unphosphorylated and phosphorylated rAQP4 have been found to be comparable that favors the reported water conductance measurements of wild type and AQP4S180D mutant structures (Mitsuma et al., 2010; Sachdeva and Singh, 2013). Hence, these MD simulations do not support the gating induced regulation of rAQP4 channel (Sachdeva and Singh, 2013). Thus, the findings of Moeller et al. (2009a) wherein reduction in water permeability of rAQP4 has been attributed to internalization of rAQP4 rather than gating, could be a possibility. Structures of full length unphosphorylated and phosphorylated rAQP4 would be further required to completely understand the effects of phosphorylation of Ser-180 on conformation of the water channel and its water permeability. Another gating event that has been proposed for AQP4 involves Ser-111 located on cytoplasmic loop B. Protein kinase A (PKA) and Protein kinase G (PKG) dependent phosphorylation of Ser-111 has been reported to increase the water permeability of AQP4 expressed in an astrocyte cell line (Gunnarson et al., 2008; Song and Gunnarson, 2012). This regulation event has been suggested to occur via gating. However, 1.8 A crystal structure of human AQP4 has been found to be in open conformation, despite the lack of a phosphate group on Ser-111 and D loop is too short to act as a gate (Ho et al., 2009). Thus, the crystal structure of human AQP4 has raised concerns regarding the proposed gating mechanism. These issues have been resolved by a recent study that determined the


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Fig. 5. Details of interactions of phosphorylated Ser-180 of rAQP4. (a) Interaction between phosphoserine-180 of loop D (magenta) with Lys-109 located on a cytoplasmic loop and Lys-311 located in the C-terminal region. 259KRR261 residues are displayed as orange spheres. (b) Top view from cytoplasmic side of phosphorylated rAQP4 showing interaction between phosphoserine-180 located on loop D (magenta) and Lys-263 lying on C-terminal helix (magenta). Loop D is located away from the channel’s cytoplasmic mouth.

extent of AQP4 gating by Ser-111 phosphorylation (Assentoft et al., 2013). There was a lack of both PKG- and PKA-induced increase in water permeability of Xenopus oocytes expressing AQP4. Moreover, there was no significant difference between the relative unit water permeability of oocytes expressing wild type AQP4 and the two mutants S111A and S111D AQP4. Since, AQP4 is a major contributor to the osmotic water permeability of the astrocyte plasma membrane (Solenov et al., 2004), thus, to determine the PKG-dependent regulatory impact on AQP4 in a more native environment, the effect of PKG activation on the osmotic water permeability has been measured in astrocytes in primary culture. No evidence could be obtained in favor of a PKG-dependent effect on the water permeability of the AQP4-containing astrocytic membrane. Molecular dynamics simulation of AQP4 with phosphorylated Ser-111 has not revealed any significant effect of the phosphorylation of Ser-111 on

the conformations of loop B or of loop D (Assentoft et al., 2013). In vivo studies using rat brain slices have not shown any detectable phosphorylation of AQP4 on Ser-111 (Assentoft et al., 2013). Thus, these findings are not in favor of phosphorylation dependent gating of AQP4 at Ser-111. 4. Yeast aquaporin gating Aquaporins in yeast have been shown to enhance their survival during rapid freezing (Tanghe et al., 2002) where extracellular water freezes faster than the intracellular water because of the higher osmolarity of the cellular content. They facilitate the rapid out flow of water out of the cell and hence less intracellular ice forms within the organism, which results in less cellular damage (Tanghe et al., 2006). A distinct feature of yeast aquaporins is that they

Fig. 6. C-terminal region of rAQP4. (a) Top view from cytoplasmic side of phosphorylated rAQP4 channel. C-terminal region (magenta) is not able to cover the mouth of water channel completely. (b) Side view of the same. Transmembrane helices are shown in blue color.


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frequently contain an extended N-terminus relative to aquaporins from other organisms. Water transport activity measurements using both proteoliposomes and spheroplasts of Pichia pastoris, have shown that full length Aqy1 is active, whereas the N-terminal truncated form of Aqy1 has dramatically increased water conductance (Fischer et al., 2009). This indicates a possible role of N-terminus in gating of the water channel. The function of this extended N-terminus has been further established with the help of X-ray structure of Aqy1 from P. pastoris (Fischer et al., 2009). The high resolution structure of Aqy1 has revealed that the water channel is closed by the N-terminus which folds in such a way that each Aqy1 protomer is intertwined with its neighboring within the tetramer via a helical bundle, which is stabilized by multiple hydrogen bonds. In the closed conformation of Aqy1, Tyr-31 is inserted into the water channel (Fig. 7). Specifically, hydrogen bond from Tyr-27 anchors the bundle of Aqy1 to the aquaporin scaffold and Pro-29 introduces a kink allowing Tyr-31 to insert into the water channel. Tyr-31 forms a hydrogen bond to a water molecule within the pore, thereby occluding the channel entrance. Pore profile of Aqy1 has revealed that the water channel narrows to as low as 0.8 A in diameter near Tyr-31, which is too small to allow the passage of water. Investigation of putative phosphorylation sites has suggested an important role of Ser-107 in regulation of Aqy1 by gating (Fischer et al., 2009). Ser-107 is situated near the pore and is involved in a network of hydrogen bonds involving Tyr-31. Water transport assays have shown a significant increase in water transport activity when Ser107 was mutated to aspartate. Further evidence in support of the role of Ser-107 has been obtained through MD simulations of Aqy1 S107D mutant which shows a widening of the pore near these residues (Fischer et al., 2009). These simulations have revealed water molecules establishing a single file water column between Pro-29, Tyr-31, Tyr-104, Leu-189, Ala-190 and Val-191, after a local rearrangement of latter three residues, which are located in helix 4. Thus, both functional data and MD simulations suggest that Ser-107 may induce the opening of Aqy1 channel upon phosphorylation. Functional assays have shown that Aqy1 has higher water transport activity when purified and reconstituted into proteoliposomes than it has in its native membrane (Fischer et al., 2009).

7

This can be explained by the unique global topology of the Aqy1 tetramer, with the N-termini intertwined in a helical bundle in a manner that is reminiscent of the arrangement found in mechanosensitive gated ion channel MscL from Mycobacterium tuberculosis. Role of mechanosensitivity in Aqy1 gating has been tested by performing MD simulations in stretched and twisted membranes. Both simulations have revealed spontaneous opening of one monomer with the pore diameter near Tyr-31 widening from 0.8 A in the crystal structure to values larger than 2 A. Moreover, the energetic barrier for the water permeation has been found to drop to less than 13 KJ mol1 compared to the control simulations in which no opening was observed. Thus, Aqy1 is suggested to be gated by a unique mechanism involving a combination of mechanosensitive gating and phosphorylation that is intimately associated with the characteristic N-terminus extension of the yeast aquaporins (Fischer et al., 2009). 5. AQP2 gating AQP2 is vasopressin regulated water channel which is expressed in the principal cells of the kidney collecting duct (Fushimi et al., 1993; Nielsen et al., 1995, 2002). In the absence of vasopressin, AQP2 is localized in a vesicle population at the subapical regions of the cell (Nielsen et al., 1995). Stimulation with vasopressin results in a predominantly apical membrane localization of AQP2 (Nielsen et al., 1995). Specifically, vasopressin initiates a signaling cascade involving increased levels of intracellular cAMP (Morel et al., 1981; Morel and Doucet, 1986), increased intracellular calcium (Chou et al., 2000), AQP2 phosphorylation (Brown et al., 2008), and subsequent AQP2 redistribution to the plasma membrane (Nielsen et al., 1995). Although mechanism of AQP2 trafficking is well studied (Brown, 2003; Valenti et al., 2005), the role of phosphorylation dependent gating of AQP2 is debatable. Various studies have identified a number of phosphorylated sites located on carboxyl terminal tail of AQP2 (Fushimi et al., 1997; Hoffert et al., 2006; Hoffert et al., 2007; Fenton et al., 2008; Hoffert et al., 2008). The polyphosphorylated region encompasses Ser-256, Ser261, Ser-264 and Ser-269. Phosphorylation of Ser-256 has been speculated to be involved in gating of the channel. However, alternative studies using different systems or similar systems have failed to agree. Protein kinase A dependent phosphorylation of Ser256 of AQP2 reconstituted in proteoliposomes has been shown to enhance the water permeability (Pf) of the channel compared with wild type AQP2 (Eto et al., 2010). A similar Pf has been observed for a S256D AQP2 mutant, whereas Pf values of wild type AQP2, S256A, S261A and S261D AQP2 mutants did not differ (Eto et al., 2010). In contrast, earlier studies of apical endosomes from rat inner medullary collecting duct cells containing AQP2 have not indicated a role for PKA-mediated phosphorylation of AQP2 (Lande et al., 1996). Studies using oocytes injected with S256D AQP2 mutant have shown similar Pf and membrane abundance compared with wild type AQP2 (Kamsteeg et al., 2000). A comprehensive analysis of relative unit Pf of AQP2 in oocytes could not provide any evidence that S256A, S261A, S264A/D and S269A/D-AQP2 mutants directly alter the water transport function of AQP2 (Moeller et al., 2009b). This study does not provide evidence in favor of phosphorylation mediated gating of AQP2 water channel. 6. AQPZ gating

Fig. 7. Aqy1 water channel is closed on the cytoplasmic side by N-terminus (magenta). Tyr-31 is inserted into the channel. Water molecules are shown as red spheres.

AQPZ is one of two members of the aquaporin family found in E. coli. It shares a high sequence similarity with the glycerol facilitator GlpF, the second member of the family. However, they vary greatly in their water permeability, with GlpF conducting water at a rate that is only about one sixth that of AQPZ (Borgnia and Agre,


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8


2001). Structural comparisons between AQPZ and GlpF have revealed the underlying mechanism of substrate selectivity in aquaporin family (Fu et al., 2000; Savage et al., 2003). The structure of AQPZ has been determined by X-ray crystallography to 2.5 A resolution (Savage et al., 2003). During MD simulation of AQPZ, a strictly conserved component of ar/R constriction site, i.e. Arg-189 has been found to flip between two distinct yet otherwise stable conformations. In one conformation, the head group of Arg-189 is oriented upwards towards the extracellular medium and the water channel is open and in another conformation, it is oriented downwards into the pore, thereby closing the channel. These observations are supported from a second X-ray structure of AQPZ to 3.2 A resolution derived from a new crystal form (Jiang et al., 2006). From electron density maps, Arg-189 has been observed to adopt the upwards-open conformation in only one monomer (Fig. 8a). In contrast, in remaining three monomers, it adopts the downwardsclosed conformation whereby the guanidinium group of Arg-189 bends over and forms hydrogen bond to carbonyl oxygen of Thr189, resulting in blocking of the ar/R site (Fig. 8bed). It has been suggested that proteineprotein interactions derived from the unique packing of tetramers within these crystals could explain the stabilization of two distinct Arg-189 conformations and hence control the water flux through the channel (Jiang et al., 2006). In this way, interactions with regulatory proteins in vivo could thus regulate water conductance. There is another report that suggests dual conformations of arginine of ar/R selectivity filter. MD simulation of rAQP4 has revealed two conformations of Arg-216, in which the dihedral angle, c4 exhibited an average value of about 100 and 170 , respectively, with the channel transiently blocked in the latter

conformation (Sachdeva and Singh, 2013). While in blocking conformation, Arg-216 interacts with backbone carbonyl oxygen of Ala-210 thereby disrupting the water flow through the channel. Similar interaction between Ala-181 and Arg-187 at ar/R site has also been noticed in electron diffraction structure of AQP0 (Han et al., 2006). Although these findings in different aquaporins suggest an elegant structural mechanism for water transport gating involving ar/R site, however its physiological significance remains speculative. 7. Gating of aquaporin-0 AQP0 is found within fiber cells of the eyes of vertebrates. It regulates water permeation across the lens cell plasma membrane and maintains homeostasis throughout the lens without becoming excessively vulnerable to external osmotic changes e.g. tears. AQP0 has also been found to participate in cellecell adhesion, where it is present in square arrays in the 11e13 nm thin membrane junctions between lens fiber cells (Costello et al., 1989). An electron diffraction structure of sheep AQP0 has been reported to 3.0 A resolution (Gonen et al., 2004) and 1.9 A resolution (Gonen et al., 2005). Packing arrangements of AQP0 tetramers in double-layered 2D crystals have been suggested to mimic those found within lens fiber cell junctions (Costello et al., 1989). Water permeability of AQP0 has been found to be 15 fold lower than that of AQP1 at pH 6.5 (Chandy et al., 1997). There is compelling evidence suggesting that decrease in external pH increases water permeability of AQP0 by three folds (Nemeth-Cahalan and Hall, 2000). Electron diffraction structure of sheep AQP0 has revealed two constriction sites along the pore

Fig. 8. Distinct conformation of ar/R sites in AQPZ. Residues making the ar/R site of AQPZ are shown for monomers A, B, C and D. Arg-189 shows upward orientation in monomer A and downward orientation in other three monomers.


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resulting in its closed conformation (Gonen et al., 2004). On the contrary, a very similar X-ray structure of bovine AQP0 at pH 10 has been shown to be dynamically open (Harries et al., 2004). Water conductance of AQP0 is low relative to other aquaporins and is altered only three fold by pH. Thus, structural changes between open and closed conformations of AQP0 are likely to be subtle. The X-ray structure (Harries et al., 2004) and electron diffraction structure (Gonen et al., 2005) of AQP0 differs significantly in the conformations of loop A, which participates in the putative junctional packing arrangement of the 2D crystals. This movement slightly displaces Met-176 and His-40 into the channel in the putative closed conformation, suggesting a gating effect induced by differences in the crystal packing. The protonation state of His-40 and Tyr-149 has been implicated in pH sensitive gating of AQP0 (Nemeth-Cahalan and Hall, 2000). However, the constriction sites observed in structures of AQP0 cannot result from the pH dependent effect as the electron diffraction structure at low pH is in closed conformation instead of being open relative to the X-ray structure at high pH. Regulation of AQP0 is also achieved by calmodulin (CaM) through a Caþ2-dependent interaction between Caþ2-CaM and the cytoplasmic C-terminal domain of AQP0 (Reichow and Gonen, 2008). CaM binds full-length AQP0 with a 2:1 (AQP0/CaM) stoichiometry. In a recent study, a pseudoatomic structure of the full length AQP0 in complex with CaM has been determined using EM (Reichow et al., 2013). The resulting AQP0-CaM structure shows the AQP0 tetramer in complex with two CaM molecules. Molecular dynamics simulations have revealed that CaM binding to the AQP0 C terminus allosterically modulates the dynamics of the pore constriction site at cytoplasmic site, resulting in channel closure. CaM regulation of AQP0 has been shown to be abolished when the gating residue, Tyr-149 at the cytoplasmic constriction site was mutated. Thus, binding of CaM at the AQP0 C terminus affects the cytoplasmic constriction gate through allosteric interactions that favor a closed conformation, thereby dynamically modulating water permeability in a Caþ2-dependent manner (Reichow et al., 2013). 8. Conclusion High resolution structures of aquaporins and MD simulations have provided useful insights into the mechanisms of aquaporin gating which are categorized as capping and pinching (Hedfalk et al., 2006). According to these mechanisms, SoPIP2;1 caps the channel by large scale rearrangement of cytoplasmic loops and place a blocking residue into the pore thereby resulting in a closed state. Similarly, in Aqy1, the channel closure is achieved upon capping of the channel by N-terminus which causes insertion of Tyr-31 into the pore. Whereas, the structural mechanisms for gating of AQP0 and AQPZ involve small conformational changes in single or few residues, which pinch on either constriction site of the pore and thereby blocking the flow of water through the channel. On the other hand, gating mechanism of AQP4 cannot be explained by either capping or pinching. MD simulations of AQP4 have not revealed any blocking residue that may get extended into the pore and restrict the passage of water (Sachdeva and Singh, 2013). Although the C-terminal region of AQP4 has been reported to move towards the cytoplasmic mouth during MD simulation, but this movement was not sufficient to cover the channel’s mouth completely and hence did not result in channel capping. Moreover, suggestions regarding mechanisms of regulation of AQP4 have been controversial. Zelenina et al. (2002) have reported that AQP4 is regulated by phosphorylation of Ser-180 via gating. However, Moeller et al. (2009a) have suggested a mechanism involving down-regulation of AQP4 upon Ser-180 phosphorylation.

9

Furthermore, findings of Mitsuma et al. (2010) and Sachdeva and Singh (2013) are not in favor of gating induced regulation of AQP4. Thus, high resolution structures of full length unphosphorylated and phosphorylated AQP4 would help to resolve the issues of gating versus trafficking based regulation of AQP4. Structural insights into aquaporin gating have shown that how the similar structural motifs are able to gate the water channel across the plant, yeast and animal kingdoms (Tornroth-Horsefield et al., 2010). Thus, basic structural themes involved in aquaporin gating have been suggested to reoccur in three major eukaryotic kingdoms (Hedfalk et al., 2006). Although, there is wealth of structural data of gating mechanisms, but along with useful insights, it has also brought a few controversies like in AQP0, AQP2 and AQP4. The mechanism of phosphorylation dependent gating of AQP2 and AQP4 is debatable. These controversies are likely to be resolved by crystal structures of full length AQP2 and AQP4 that will help to determine the role of C-terminus in gating at the atomic level. Acknowledgments Authors are thankful to the Council of Scientific and Industrial Research (CSIR), Government of India for financial assistance. References Alleva, K., Niemietz, C.M., Sutka, M., Maurel, C., Parisi, M., Tyerman, S.D., Amodeo, G., 2006. Plasma membrane of beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations. J. Exp. Bot. 57, 609e621. Amiry-Moghaddam, M., Ottersen, O.P., 2003. The molecular basis of water transport in the brain. Nat. Rev. Neurosci. 4, 991e1001. Assentoft, M., Kaptan, S., Fenton, R.A., Hua, S.Z., de Groot, B.L., MacAulay, N., 2013. Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating. Glia 61, 1101e1112. Azad, A.K., Katsuhara, M., Sawa, Y., Ishikawa, T., Shibata, H., 2008. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation. Plant Cell Physiol. 49, 1196e1208. Borgnia, M., Nielsen, S., Engel, A., Agre, P., 1999. Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425e458. Borgnia, M.J., Agre, P., 2001. Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli. Proc. Natl. Acad. Sci. USA 98, 2888e2893. Brown, D., 2003. The ins and outs of aquaporin-2 trafficking. Am. J. Physiol. Ren. Physiol. 284, F893eF901. Brown, D., Hasler, U., Nunes, P., Bouley, R., Lu, H.A., 2008. Phosphorylation events and the modulation of aquaporin 2 cell surface expression. Curr. Opin. Nephrol. Hypertens. 17, 491e498. Chandy, G., Zampighi, G.A., Kreman, M., Hall, J.E., 1997. Comparison of the water transporting properties of MIP and AQP1. J. Membr. Biol. 159, 29e39. Chen, Y.C., Cadnapaphornchai, M.A., Schrier, R.W., 2005. Clinical update on renal aquaporins. Biol. Cell. 97, 357e371. Chou, C.L., Yip, K.P., Michea, L., Kador, K., Ferraris, J.D., Wade, J.B., Knepper, M.A., 2000. Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Caþ2 stores and calmodulin. J. Biol. Chem. 275, 36839e36846. Conner, M.T., Conner, A.C., Brown, J.E., Bill, R.M., 2010. Membrane trafficking of aquaporin 1 is mediated by protein kinase C via microtubules and regulated by tonicity. Biochemistry 49, 821e823. Costello, M.J., McIntosh, T.J., Robertson, J.D., 1989. Distribution of gap junctions and square array junctions in the mammalian lens. Invest. Ophthalmol. Vis. Sci. 30, 975e989. de Groot, B.L., Frigato, T., Helms, V., Grubmuller, H., 2003. The mechanism of proton exclusion in the aquaporin-1 water channel. J. Mol. Biol. 333, 279e293. de Groot, B.L., Grubmuller, H., 2005. The dynamics and energetics of water permeation and proton exclusion in aquaporins. Curr. Opin. Struct. Biol. 15, 176e183. Eto, K., Noda, Y., Horikawa, S., Uchida, S., Sasaki, S., 2010. Phosphorylation of aquaporin-2 regulates its water permeability. J. Biol. Chem. 285, 40777e40784. Fenton, R.A., Moeller, H.B., Hoffert, J.D., Yu, M.J., Nielsen, S., Knepper, M.A., 2008. Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin. Proc. Natl. Acad. Sci. USA 105, 3134e3139. Fischer, G., Kosinska-Eriksson, U., Aponte-Santamaria, C., Palmgren, M., Geijer, C., Hedfalk, K., Hohmann, S., de Groot, B.L., Neutze, R., Lindkvist-Petersson, K., 2009. Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism. PLoS Biol. 7, e1000130.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Q1 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65


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Fu, D., Libson, A., Miercke, L.J., Weitzman, C., Nollert, P., Krucinski, J., Stroud, R.M., 2000. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290, 481e486. Fushimi, K., Sasaki, S., Marumo, F., 1997. Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel. J. Biol. Chem. 272, 14800e14804. Fushimi, K., Uchida, S., Hara, Y., Hirata, Y., Marumo, F., Sasaki, S., 1993. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 361, 549e552. Gonen, T., Cheng, Y., Sliz, P., Hiroaki, Y., Fujiyoshi, Y., Harrison, S.C., Walz, T., 2005. Lipid-protein interactions in double-layered two dimensional AQP0 crystals. Nature 438, 633e638. Gonen, T., Sliz, P., Kistler, J., Cheng, Y., Walz, T., 2004. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429, 193e197. Gunnarson, E., Axehult, G., Baturina, G., Zelenin, S., Zelenina, M., Aperia, A., 2005. Lead induces increased water permeability in astrocytes expressing aquaporin 4. Neuroscience 136, 105e114. Gunnarson, E., Zelenina, M., Axehult, G., Song, Y., Bondar, A., Krieger, P., Brismar, H., Zelenin, S., Aperia, A., 2008. Identification of a molecular target for glutamate regulation of astrocyte water permeability. Glia 56, 587e596. Han, B.G., Guliaev, A.B., Walian, P.J., Jap, B.K., 2006. Water transport in AQP0 aquaporin: molecular dynamics studies. J. Mol. Biol. 360, 285e296. Harries, W.E., Akhavan, D., Miercke, L.J., Khademi, S., Stroud, R.M., 2004. The channel architecture of aquaporin 0 at a 2.2-A resolution. Proc. Natl. Acad. Sci. USA 101, 14045e14050. Hedfalk, K., Tornroth-Horsefield, S., Nyblom, M., Johanson, U., Kjellbom, P., Neutze, R., 2006. Aquaporin gating. Curr. Opin. Struct. Biol. 16, 447e456. Heller, K.B., Lin, E.C., Wilson, T.H., 1980. Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J. Bacteriol. 144, 274e278. Hiroaki, Y., Tani, K., Kamegawa, A., Gyobu, N., Nishikawa, K., Suzuki, H., Walz, T., Sasaki, S., Mitsuoka, K., Kimura, K., Mizoguchi, A., Fujiyoshi, Y., 2006. Implications of the aquaporin-4 structure on array formation and cell adhesion. J. Mol. Biol. 355, 628e639. Ho, J.D., Yeh, R., Sandstrom, A., Chorny, I., Harries, W.E., Robbins, R.A., Miercke, L.J., Stroud, R.M., 2009. Crystal structure of human aquaporin 4 at 1.8 Å and its mechanism of conductance. Proc. Natl. Acad. Sci. USA 106, 7437e7442. Hoffert, J.D., Fenton, R.A., Moeller, H.B., Simons, B., Tchapyjnikov, D., McDill, B.W., Yu, M.J., Pisitkun, T., Chen, F., Knepper, M.A., 2008. Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2. J. Biol. Chem. 283, 24617e24627. Hoffert, J.D., Nielsen, J., Yu, M.J., Pisitkun, T., Schleicher, S.M., Nielsen, S., Knepper, M.A., 2007. Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct. Am. J. Physiol. Ren. Physiol. 292, F691eF700. Hoffert, J.D., Pisitkun, T., Wang, G., Shen, R.F., Knepper, M.A., 2006. Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin2 phosphorylation at two sites. Proc. Natl. Acad. Sci. USA 103, 7159e7164. Horsefield, R., Norden, K., Fellert, M., Backmark, A., Tornroth-Horsefield, S., Terwisscha van Scheltinga, A.C., Kvassman, J., Kjellbom, P., Johanson, U., Neutze, R., 2008. High-resolution x-ray structure of human aquaporin 5. Proc. Natl. Acad. Sci. USA 105, 13327e13332. Jiang, J., Daniels, B.V., Fu, D., 2006. Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel. J. Biol. Chem. 281, 454e460. Johanson, U., Karlsson, M., Johansson, I., Gustavsson, S., Sjovall, S., Fraysse, L., Weig, A.R., Kjellbom, P., 2001. The complete set of genes encoding major intrinsic proteins in arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol. 126, 1358e1369. Johansson, I., Karlsson, M., Shukla, V.K., Chrispeels, M.J., Larsson, C., Kjellbom, P., 1998. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. Plant Cell. 10, 451e459. Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B., Baraban, J.M., Agre, P., 1994b. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc. Natl. Acad. Sci. U S A 91, 13052e13056. Jung, J.S., Preston, G.M., Smith, B.L., Guggino, W.B., Agre, P., 1994a. Molecular structure of the water channel through aquaporin CHIP. The hourglass model. J. Biol. Chem. 269, 14648e14654. Kamsteeg, E.J., Heijnen, I., van Os, C.H., Deen, P.M., 2000. The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers. J. Cell. Biol. 151, 919e930. King, L.S., Kozono, D., Agre, P., 2004. From structure to disease: the evolving tale of aquaporin biology. Nat. Rev. Mol. Cell. Biol. 5, 687e698. Kuriyama, H., Shimomura, I., Kishida, K., Kondo, H., Furuyama, N., Nishizawa, H., Maeda, N., Matsuda, M., Nagaretani, H., Kihara, S., Nakamura, T., Tochino, Y., Funahashi, T., Matsuzawa, Y., 2002. Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes 51, 2915e2921. Lande, M.B., Jo, I., Zeidel, M.L., Somers, M., Harris Jr., H.W., 1996. Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles. J. Biol. Chem. 271, 5552e5557. 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