Experimental and Simulation Studies of Aquaporin 0 Water

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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Experimental and Simulation Studies of Aquaporin 0 Water Permeability and Regulation James E. Hall,*,† J. Alfredo Freites,‡ and Douglas J. Tobias*,‡ Department of Physiology and Biophysics and ‡Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States

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ABSTRACT: We begin with the history of aquaporin zero (AQP0), the most prevalent membrane protein in the eye lens, from the early days when AQP0 was a protein of unknown function known as Major Intrinsic Protein 26. We progress through its joining the aquaporin family as a water channel in its own right and discuss how regulation of its water permeability by pH and calcium came to be discovered experimentally and linked to lens homeostasis and development. We review the development of molecular dynamics (MD) simulations of lipid bilayers and membrane proteins, including aquaporins, with an emphasis on simulation studies that have elucidated the mechanisms of water conduction, selectivity, and proton exclusion by aquaporins in general. We also review experimental and theoretical progress toward understanding why mammalian AQP0 has a lower water permeability than other aquaporins and the evolution of our present understanding of how its water permeability is regulated by pH and calcium. Finally, we discuss how MD simulations have elucidated the nature of lipid interactions with AQP0.

CONTENTS 1. Introduction 1.1. Aquaporin History: Early Days 1.2. MIP 26 Becomes Aquaporin 0 (Sort Of) 1.3. Ubiquity of Aquaporins and AQP0 Tissue Specificity 1.4. AQP1 and AQP0 Structure Determination 1.5. Mechanistic Aspects of AQP Gating and Selectivity Inferred from High-Resolution Structures 1.6. AQP0 Water Permeability 2. Role of AQP0 in Lens Physiology and Development 3. Experimental Studies of AQP0 Regulation 3.1. Regulation Mechanisms Constrained by Energy 3.2. Regulation by pH 3.3. Regulation by Ca2+ 3.4. Cooperativity 4. Molecular Dynamics Simulations of AQPs 4.1. Molecular Dynamics Simulations of Membranes and Membrane Proteins: A Brief History 4.2. Mechanistic Details of AQP Water Conduction, Gating, and Selectivity Revealed by MD Simulation Studies 4.3. Making Contact with Experiment: Calculating pf from MD Simulations 5. Molecular Dynamics Simulations of AQP0 5.1. Simulation Studies of Nonjunctional AQP0 Permeability and Gating

5.2. Simulation Studies Comparing Junctional and Nonjunctional AQP0 5.3. Simulation Studies of AQP0 Permeability Regulation by Ca2+ and pH 5.4. Simulation Studies of AQP0−Lipid Interactions 6. Conclusions and Future Directions Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION 1.1. Aquaporin History: Early Days

Aquaporin zero (AQP0) came to the attention of researchers as MIP 26, MIP being short for Major Intrinsic Protein, and 26 being the location where the protein ran on gels at 26 kDas.1,2 The original name was appropriate. MIP 26 was more than 60% of the membrane protein of the lens of the eye, and simply by grinding up lenses and isolating the membrane fraction, one could get a preparation of nearly pure protein.

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Special Issue: Biomembrane Structure, Dynamics, and Reactions

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Received: February 15, 2019

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DOI: 10.1021/acs.chemrev.9b00106 Chem. Rev. XXXX, XXX, XXX−XXX

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bovine AQP0.18 There are more variants of aquaporins in plants than in vertebrates, and many of these variants show Pf regulation by both pH and calcium.19 While this review is focused on mammalian AQP0, found almost exclusively in the lens of the eye (but with traces in testes20 and liver21), AQP0 is not the only aquaporin with regulated Pf. Readers interested in comparing mechanisms of regulation across multiple different aquaporins will want to consult an excellent review by Törnroth-Horsefield et al.22

The lens was a convenient tissue for microscopists, and easy access to the lens allowed high-resolution structural studies, which revealed junctional structures of two kinds: hexagonal arrays and square arrays, both of which were judged to be a species of gap junction.3 In 1984, Gorin et al. cloned MIP 26 and concluded that its deduced structure was consistent with it being a gap junction protein.4 This supported the erroneous belief that MIP was an ion channel and, indeed, reconstitution in lipid bilayers seemed to confirm that notion.5 But, this was a short-lived idea. In 1991, Greg Preston and Peter Agre sequenced a 28 kDa protein found in red blood cells and kidney proximal tubule.6 Based on its similarity to MIP 26, they gave it the name CHIP 28 (short for CHannel-like Intrinsic Protein). The thinking was that it might be an ion channel. But, as it turned out, it was much more than that. In 1992, Greg Preston, Tiziana Caroll, William Guggino, and Peter Agre published the archetypical aquaporin paper showing that CHIP 28 was in fact the long sought mercury-sensitive water permeation pathway in red blood cells.7 They made the important observation that MIP 26 and CHIP 28 had about 42% sequence identity, likely had the same membrane topology, and differed most profoundly in the C-terminal region, later shown to be the critical region for phosphorylation and regulation.7 In retrospect, there was a clear hint in 1975 that the protein family that later became the aquaporins was a major mediator of water permeability, but everyone missed the connections among a number of observations from different fields. The crucial link that everyone missed was the proportionality between antidiuretic hormone−vasopressin induced increase in the water permeability of toad bladder and the increase in the number of freeze fracture particles, in square arrays, in the basolateral membrane.8 We now know that the protein responsible for both the increase in water permeability and the increase in the number of freeze fracture particles is aquaporin 2.9 But, we could have known in 1975 with a more careful reading of the literature and a perhaps a little luck!

1.4. AQP1 and AQP0 Structure Determination

The presence of large amounts of easily isolated and purified AQP0 in the lens would seem to have made the crystallization of AQP0 for high resolution structural studies a simple matter. But, this did not prove to be as straightforward as initially hoped, largely because initial crystallization methods yielded crystals that did not diffract well enough for structure determination. AQP1 turned out to be easier to crystallize and, hence, its structure was determined well before that of AQP0. For a survey of all AQP structures determined up to 2006, the reader is referred to the excellent review by Gonen and Walz.23 The first “structures” of AQP1, projection structures24,25 and a three-dimensional reconstruction26 obtained by electron microscopy (EM) of two-dimensional crystals with 12−16 Å resolution, showed that the functional unit of AQP1 is a tetramer and that the helices in each monomer were oriented normal to the membrane plane. In 1997, three AQP1 structures determined to 6−7 Å resolution by electron crystallography were reported; these structures revealed that, within each monomer, six tilted transmembrane helices pack into a right-handed bundle with pseudo-twofold symmetry that surrounds a central density.27−29 A subsequent 4.5 Å resolution structure showed that two short helical segments (referred to by researchers in the aquaporin field as “half helices”) enclosed by the transmembrane helix bundle meet at the highly conserved Asn-Pro-Ala (NPA) motifs in the middle of the membrane, and hinted strongly that the water conducting pore runs through the center of the monomer.30 Thus, this structure essentially confirmed the “hourglass model” of AQP1 that had been proposed prior to the availability of three-dimensional structures.31 The water conduction pathway in AQP1 was clearly delineated in the first atomically detailed structure, determined to 3.8 Å resolution from electron crystallographic data and reported in 2000.32 The water-conducting pore consists of extracellular and cytoplasmic vestibules connected by a narrow pore that serves as the selectivity filter. The exquisite selectivity of most AQPs for water33,34 was partially explained by the 3 Å diameter of the narrowest section of the AQP1 pore. In addition to confirming the AQP1 fold in the 3.8 Å structure solved by Murata et al.,32 a 2.2 Å resolution structure determined by Sui et al. using X-ray crystallography provided the first glimpse of water molecules in the water-conducting pore of AQP1.35 The four water molecules resolved in the structure were in the selectivity filter, the extracellular side of which is also referred to as the ar/R constriction site, because it is formed by a cluster of highly conserved aromatic and arginine residues. In 2004, Gonen and colleagues solved the structure of closed AQP0 in the junctional form using electron crystallography of two-dimensional crystals.36 This was the first AQP0 structure solved to high resolution at 3 Å. It was this junctional form

1.2. MIP 26 Becomes Aquaporin 0 (Sort Of)

By an accident of fate, investigators first looked at mammalian aquaporin 0 (AQP0), which has a much lower water permeability, Pf, than aquaporin 1 (AQP1) or fish AQP0. (Why fish AQP0s have such a high Pf and mammalian AQP0s such a low one is not yet known.) The permeability of bovine AQP0 was first reported by several groups, most notably, in a paper by Mulders et al. 1995,10 but also in meeting abstracts by Kusmerick et al.11 and Zampighi et al.12 Kushmerick et al. subsequently followed up their abstract with a full publication.13 Some groups even concluded that MIP 26 was, in fact, not a water channel.14 But, it eventually became clear that even mammalian AQP0, despite its low Pf, is in fact a bona f ide water channel. In 1993, Agre, Sasaki, and Chrispeels proposed that MIP 26related proteins be referred to as “aquaporins”.15 Furthermore, although AQP1 was the first protein of the family shown to be a water channel, MIP 26 was the first to be sequenced, and comparison with this sequence led to the identification of CHIP 28/AQP1. Consequently, because the number one was already taken, MIP 26 became aquaporin 0. 1.3. Ubiquity of Aquaporins and AQP0 Tissue Specificity

Aquaporins are an ancient protein family. There are aquaporins in bacteria, vertebrates, invertebrates,16 and plants.17 In fact, the plant aquaporin, nodulin 26, shows 35% homology to B


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Figure 1. Tetramer and monomer structure of bovine AQP0 (bAQP0). (Upper Left) Cartoon of the bAQP0 tetramer looking down the transmembrane axis from the extracellular side of the protein. Yellow and blue indicate structures derived from each of the two gene-duplicated portions of the primary sequence. (Upper Right) Cartoon showing the same view as in Upper Left with each monomer shown in a different representation. (Lower) Cartoon of an bAQP0 monomer in a side view, with the uppermost extracellular side in crossed eye stereo. Water oxygens are shown as red spheres. All images were made with PYMOL (DeLano Scientific, San Carlos, CA). Adapted with permission from ref 37. Copyright 2004 National Academy of Sciences, United States.

simulations, (reviewed in Sections 4 and 5). These simulations have provided many additional important insights into the mechanisms of the water permeation and selectivity of aquaporins beyond those that could be inferred from static structures as well as into the mechanism of the regulation of AQP0 by Ca2+.

revealed in freeze-fracture microscopy that had led earlier investigators to believe that MIP, now called AQP0, formed gap junctions. Shortly after the publication of the closed, junctional form of AQP0 was published, Harries et al. reported a 2.2 Å resolution structure of the open, nonjunctional form determined by X-ray crystallography (Figure 1).37 Subsequently, the resolution of the junctional form of AQP0 was improved to 1.9 Å.38 Similar to other AQPs, the highresolution AQP0 structures showed that the overall fold of AQP0 is very similar to that of AQP1. However, the AQP0 structures revealed two additional structural features that are not found in other AQPs, to be described below in Subsection 1.5, that impede water passage through the water-conducting pores. The structural work reviewed in this Subsection, which took place in parallel with multiple functional studies, provided a basis for understanding the water permeability of aquaporins (see Subsection 1.5) and its regulation by a variety of mechanisms (see Section 3). The availability of high-resolution structures of AQP1 and AQP0, as well as that of the aquaglyceroporin GlpF,39 also opened the door for computational modelers to use all-atom molecular dynamics (MD)

1.5. Mechanistic Aspects of AQP Gating and Selectivity Inferred from High-Resolution Structures

The functional unit of AQPs is a tetramer (Figure 1, upper left) in which each monomer contains its own waterconducting pore (Figure 1, bottom panels). In the monomer, the six transmembrane helices and the NPA motifs at the junctions between the two short helices HB and HE are widely conserved structural features (Figure 2). The two short helices that meet at the NPA motifs are oriented such that their dipoles are pointed toward the side chain of the Asn residue in the NPA motif. Although their structure did not contain any resolvable water molecules, Murata et al. posited that the lack of AQP1 permeability to protons is due to a break in the connectivity of a water single file in the middle of the conducting pore, effected by the positive electrostatic potential generated at the ends of the pore-lining helices and focused on C


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Y149, F75, and H66 located on the cytoplasmic side of the narrow part of the pore (Figure 2).36 The high B factor of the side chain of Y149 suggested that it could gate water conduction by swinging in and out of the pore.36 This suggestion has been confirmed by MD simulations reviewed in Section 5. The second unique feature of AQP0 is the presence of the pore-lining residue Y23, which is located on the extracellular side of the pore between the CS I and the NPA motifs (Figure 2); the corresponding residue in AQP1 is F24, which lacks a hydrogen-bonding moiety. In an AQP0 structure,38 the side chain of Y23 is seen to disrupt the continuous hydrogen-bonded chain of water molecules in the narrow part of the pore, thus creating a “phenolic barrier” to water conduction.23,38 MD simulations reviewed in Section 5 confirm that Y23 plays a significant role in reducing the water permeability of AQP0 relative to that of AQP1 and other AQPs. 1.6. AQP0 Water Permeability

Figure 2. Crystallographic structure of the bovine AQP0 monomer.37 Transmembrane helices are labeled H1 through H6, and loops connecting transmembrane helices are denoted loop A through loop E. Two half helices, HB and HE, emanate from the corresponding connecting loops (loop B and loop E) and meet at the NPA motifs (locations of the Asn side chains are denoted by asterisks). The Cterminal helix labeled CBD is the calmodulin-binding domain. Also shown are amino acid residue side chains key to water permeation regulation (R187 and H172 from CS I, Y23, and Y149 from CS II, and the pH-sensing H40), as well as crystallographic water oxygen atoms in the channel pathway. The figure was rendered with VMD.42 The protein is shown in secondary structure representation and colored by residue number using a BGR color scale (from the Nterminus to the C-terminus). The water oxygens and amino acid residue side chains are colored by atom (carbon, silver; oxygen, red; nitrogen, blue; hydrogen, white). Missing residues in loop A were modeled using the bovine AQP0 electron crystallographic structure.38 Hydrogen atoms were modeled using VMD.42

Here, we comment briefly on the magnitude of the water permeability of AQP0. Many researchers have demonstrated that the water permeability of AQP0 is much smaller that that of AQP1.10,43,44 This is certainly true of mammalian AQP0s, particularly human and bovine. Although the values vary somewhat depending on the details of the measurement, the single-channel permeability (pf) of mammalian AQP1 has generally been reported to be roughly 2 orders of magnitude higher than that of mammalian AQP0.10,43,44 For example, Chandy et al. reported pf values of 1.2 × 10−14 and 2.8 × 10−16 cm3/s for AQP1 and AQP0, respectively.43 However, the fish AQP0s, MIPfun from killifish (Fundulus heteroclitus), Aqp0a and at least one form of Aqp0b from zebrafish (Danio rerio), and AQP0s from salmon have permeability values much higher than those of mammalian AQP0.45−47 The water permeability of single monomers of AQP1, CHIP 28, was estimated in 1992 by two groups using red blood cells and protein reconstituted from red cells into liposomes.33,48 Both groups found the AQP1 single monomer pf to be about 10−13 cm3/s (note: herein we use Pf to denote whole cell water permeability in units of μm/s or cm/s and pf to denote singlechannel water permeability in units of cm3/s; the latter is obtainable from the former if the expression level of the water channels in the permeability assays is known). Estimates of the single-channel pf of AQP0 had to wait until 1997. Two groups estimated the permeability using very different methods. Yang and Verkman estimated the number of AQP0 molecules in their oocyte preparations using quantitative immunology.44 They estimated the pf to be 2.5 × 10−15 cm3/sec. Chandy et al. counted the number of particles in the plasma membrane of oocytes expressing AQP0 for different expression levels and deduced a pf of 2.8 × 10−16 cm3/sec.43 This value, a factor of 10 less than the value reported by Yang and Verkman, is likely closer to the true value owing to a correction made by Chandy et al. that is not typically made in calculating Pf in oocyte swelling assays. It turns out that the true surface area of an oocyte is about 9 times larger than the geometric surface area. This correction reduces the Pf calculated from a given rate of change of volume induced by an osmotic challenge by a factor of 9.

Asn side chains in the NPA motifs.32 The interruption of the chain of hydrogen bonds in the pore was expected to shut down proton transfer via Grotthuss proton hopping across the middle of the pore. Indeed, such a disruption in the hydrogen bonds between water molecules in the pore were observed in early MD simulations of AQP1 and GlpF, which did not include explicit protons.40,41 However, as will be discussed in Subsection 4.1, this mechanistic picture of proton exclusion had to be revised once MD simulations of AQPs containing explicit protons in their pores were carried out. As mentioned above, the narrowest section of the selectivity filter of the water pore of AQP1 is the highly conserved ar/R constriction site. In AQP1 it is formed by a cluster of the side chains of R197, H182, and F58, while in AQP0 it is formed by R187, F48, and H172. The diameter of this constriction, ∼3 Å, is barely large enough to permit the passage of water molecules, and too narrow to permit the passage of larger solutes. Furthermore, the lack of an arrangement of polar moieties in the selectivity filter capable of removing water molecules from ion hydration shells impedes the conduction of ions. In addition to the ar/R constriction site (also called the first constriction site or CS I in AQP0) found in many other AQPs, AQP0 has two additional and unique structural features that impede water conduction through its pores. One is a second constriction site (CS II), which is formed by the side chains of D


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Figure 3. Effect of pH on AQP0 osmotic water permeability, Pf. (A) Decreasing pH from 7.5 to 6.5 increases the water permeability of AQP0injected oocytes by a factor of 3.4 ± 0.4 but has no effect on the water permeability of AQP1 or uninjected oocytes. (B) Titration curve of the relative permeabilities of AQP0, AQP1, and uninjected oocytes from pH 8.0 to 5.5. Only AQP0 exhibits a pH-dependent water permeability. (C) Relative permeability of AQP0 at pH 6.5 is not altered by expression level. To obtain a wider range of water permeabilities, 5 or 10 ng of AQP0 cRNA were injected. Expression level, as monitored by permeability at pH 7.5, is shown on the abscissa. The ordinate is the relative permeability at pH 6.5 calculated as (Pf at pH 6.5 − PfUI)/(Pf at pH 7.5 − PfUI), where PfUI is the osmotic water permeability of uninjected oocytes. (D) Lowering expression level does not result in pH sensitivity of either AQP1 or AQP4. Lowered expression levels of AQP1 and AQP4 were achieved by injecting different volumes of diluted cRNA, resulting in similar water permeabilities to those induced by injection of 10 ng of AQP0 cRNA. Unless otherwise indicated, each data point is the average of nine measurements (three different batches of oocytes, three oocytes from each batch). Adapted with permission from ref 63. Copyright 2000 American Society for Biochemistry and Molecular Biology.

2. ROLE OF AQP0 IN LENS PHYSIOLOGY AND DEVELOPMENT

3. EXPERIMENTAL STUDIES OF AQP0 REGULATION 3.1. Regulation Mechanisms Constrained by Energy

Functional AQP0 is essential for a clear lens and proper lens development. AQP0 was first implicated in cataract while it was still MIP. Adrey Muggleton-Harris identified cataractproducing mutants Lop and later cataract Fraser.49 A few years later, Alan Shiels and coworkers showed that the defective gene generating the cataract was MIP.50,51 Still later, Kalman et al. showed that expressing both normal AQP0 and the AQP0LTR mutant responsible for cataract in mice together in a transgenic mouse reduced the severity of the cataract but did not eliminate it altogether.52 In the same paper, Kalman et al. found that coexpressing AQP0 and AQP0-LTR in Xenopus oocytes eliminated the calcium regulation of water permeability.52 This suggested for the first time that both water permeability and its regulation by Ca2+ were essential for maintaining lens clarity. Knockdown and knockout experiments in zebrafish confirmed that intact functional AQP0 is essential for lens clarity. Initially, morpholino knockdown experiments showed that both forms of AQP0 found in the zebrafish (Aqp0a and Aqp0b) are essential for lens clarity.53 Additional zebrafish experiments showed that, not only is Aqp0a essential, but Ca2+ regulation of Aqp0a water permeability is as well.54 (As a side note, Aqp0b seems to function primarily as an adhesive protein.53,54) Subsequently, CRISPR-Cas9 knockouts of Aqp0a and Aqp0b in zebrafish provided more detail on how loss of either or both aquaporins affects lens development and clarity and confirmed the morpholino findings that both proteins are essential for proper development and a clear lens.55

One type of regulation of water permeability by aquaporins was actually well-known before aquaporins were identified. This was the antidiuretic hormone (ADH)-controlled water permeability increase in the distal tubule and collecting duct of the kidney, which promoted reabsorption of water and production of a concentrated urine. The water permeability increase was strictly proportional to the increase of the number and density of “square arrays” resembling the square arrays found in the eye lens, which were formed by MIP, later renamed AQP0.8 Later, it was shown that the ADH-controlled mechanism resulted in fusion with and removal from the apical plasma membrane of long cylindrical vesicles containing aquaporin 2.9 This increased or decreased the Pf of the tubules so as to promote formation of a concentrated urine when Pf was high or a dilute urine when Pf was low. This mechanism places a heavy demand on metabolic energy. This is not a problem in the kidney, which gets as much of the cardiac output as it needs. However, it is clearly not appropriate in the lens, which has no blood vessels and no claim on cardiac output whatsoever. Thus, an important question is how does the lens regulate the Pf of AQP0? 3.2. Regulation by pH

Based on energetic considerations, several laboratories considered that changes in ionic composition might alter the Pf of AQP0 in the lens. Two ions, Ca2+ and protons, were prime candidates because pH decreases toward the center of the lens.56,57 and the concentration of intracellular Ca2+ increases toward the lens center.58 Moreover disturbances in Ca2+ concentration are associated with cataract.59−61 E


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Two laboratories investigated the effects of pH on the permeability of AQP0 in Xenopus oocytes. Zeuthen and Klaerke found no dependence on pH of AQP0 Pf,62 but Németh-Cahalan and Hall did find a significant change in Pf with pH (Figure 3).63 Note that the titration curve is biphasic and that, if one tests the Pf at neutral pH and pH 4.5, the values are about the same, and one might conclude that the Pf did not depend on pH at all (Figure 3B). The water permeabilities of uninjected oocytes and of AQP1 and AQP4 do not depend on pH and serve as negative controls. Treatment of exposed histidines with diethylpyrocarbonate (DEPC) selectively alters histidine residues.64 Németh-Cahalan et al. showed that treating AQP0-injected oocytes with DEPC eliminates pH sensitivity of the water permeability.63 This finding strongly indicated that one or more external histidines mediated the pH sensitivity of AQP0. This suggestion was confirmed using single residue mutations.65 Németh-Cahalan et al. replaced histidines in AQP0 and in other AQP analogues as well (Figure 4). Figure 4B shows that the histidines in loop A (Figure 2) have a profound effect on the Pf of both AQP0 and MIPfun (the AQP0 of the killifish, F. heteroclitus). While acid pH increases the Pf of bovine (and human) AQP0, basic pH increases the Pf of MIPfun (Figure 4A). But, the sensitivity of both isoforms can be altered by histidine mutation, either positional or compositional, as Figure 4B shows. When H40 in AQP0 is mutated to Cys, the Pf of the mutant form of AQP0 increases with basic pH, not acidic pH, as the Pf of wild-type (WT) AQP0 does (Figure 4B, right panel). Moreover, shifting the position of H39 in MIPfun to the same position the His in WT AQP0 occupies converts MIPfun Pf from a basic sensitive protein to an acid sensitive one (Figure 4B, left panel). These experiments clearly establish the role of histidines and their locations in the pH control of Pf in AQP0s. Németh-Cahalan et al. also introduced histidines at different positions into AQP1, which has a much higher Pf than bAQP0, but is normally not sensitive to either basic or acidic pH.65 Doing so conferred either acid or basic sensitivity of Pf depending on the location of the introduced histidines. Note that because of the higher magnitude of the Pf of AQP1, the change in Pf induced by acid or basic pH changes was correspondingly higher in the pH-sensitive mutants. Bovine AQP0 and MIPfun are not the only AQP0s with pHsensitive water permeabilities. Chauvigné et al. investigated the pH sensitivity of a wide range of fish AQP0s and found acidic pH sensitive, basic pH sensitive, and pH insensitive paralogues.47 Tetraploid salmon have four paralogues of AQP0, and these exhibit multiple forms of pH sensitivity. The two AQP0s found in zebrafish have different pH sensitivities (Figure 5). Recently, Saboe et al. have investigated the pH dependence of AQP0 reconstituted into liposomes.66 In contrast with Németh-Cahalan et al.,65 Saboe et al. found that the pH increases with basic pH, not with acid pH (Figure 6). NémethCahalan et al. and Saboe et al. used different experimental systems, oocyte expression and reconstituted liposomes, respectively, so the differences between the systems could account for the differences in measured pH sensitivity, but exactly how is not clear. One possibility is that the oocyte system has accessory proteins such as calmodulin, AKAP2, and PKA.67 All of these are known to alter the Pf of AQP0 in expression systems.65,67 A second possibility might be the

Figure 4. Role of histidines in loop A on bAQP0 and MIPfun osmotic water permeability. Control conditions were pH 7.5 and 1.8 mM Ca2+. Ion substitutions were made one at a time relative to control. (A) The permeability of bAQP0-injected oocytes increases by a factor of 1.8 at pH 6.5 and by a factor of 2.1 with no added Ca2+. MIPfuninjected oocytes have a water permeability 11 times higher than that of oocytes injected with the same quantity of AQP0 cRNA under control conditions (pH 7.5, 1.8 mM Ca2+). The permeability of MIPfun-injected oocytes increases by a factor of 2.2 at pH 8.5 and by a factor of 1.9 with no added Ca2+. (B) Moving the H39 to the position 40 restores the acid pH sensitivity, but replacing H43 does not affect the pH sensitivity. Acid pH has no effect on the water permeability of bAQP0/H40C-injected oocytes, but no added Ca2+ or alkaline pH increases the permeability by a factor of 1.8 and 1.9, respectively. Note the different scales for bAQP0 and MIPfun. Adapted with permission from ref 65. Copyright 2004 Rockefeller University Press.

influence of the lipid system used for reconstitution by Saboe et al. and the details of the reconstitution protocol. Tong et al. found that both the single-channel pf of AQP0 and the degree of its pH sensitivity depended on lipid composition.68 Figure 7 shows that increasing the percent of cholesterol or sphingomyelin in the membrane used for reconstitution lowers the permeability of AQP0. It is possibly significant that Tong et al. used purified defined acyl chain lipids in their reconstitution systems. Tong et al. also studied the pH dependence of AQP0 pf, the magnitude of which they found to be dependent on lipid composition (Figure 8). They measured an increase in singlechannel permeability with acidic pH, in qualitative agreement with the results of Németh-Cahalan et al.63 The results of Tong et al. suggest an additional explanation for the results of Saboe et al., namely, that lipid composition, and especially the nature of the acyl chains, may shift the pH dependence. Tong et al’s results clearly establish a role for lipid composition in determining the pf of not only AQP0, but also of AQP4 and AQP1. They suggest that the degree of hydrophobic mismatch F


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Figure 5. Both zebrafish aqp0a and 0b paralogues encode functional water channels. Osmotic water permeability (Pf) of X. laevis oocytes injected with water or cRNA encoding zebrafish wild-type Aqp0a or Aqp0b or the Aqp0b-G19S mutant. The Pf was calculated using an estimated surface area of 9× the geometric area. Oocytes were exposed to different pH conditions before and during the swelling assays. Data are the mean ± SEM (n = 8−12 oocytes per treatment) of 4 independent experiments. Significant differences (*P, 0.05; **P, 0.01; ***P, 0.001) for each construct at the 3 pH values are indicated. The bracket indicates significant differences with respect to waterinjected oocytes. NS, not significant. Adapted with permission from ref 47. Copyright 2015 the Federation of American Societies for Experimental Biology.

Figure 7. Single-channel water permeabilities for AQP0 in bilayers composed of POPC:POPG, POPC:POPG:cholesterol, and SM:DPPG:cholesterol obtained at pH 7.5. Adapted with permission from ref 68. Copyright 2013 Elsevier.

Figure 8. Single-channel water permeabilities for AQP0 in bilayers composed of POPC:POPG:cholesterol and SM:DPPG:cholesterol at pH 7.5 and 6.5. Reproduced with permission from ref 68. Copyright 2013 Elsevier.

Figure 6. Osmotic water permeability of AQP0 at pH 6.5 and 7.5. Incorporation of AQP0 purified from sheep lenses (native) and recombinant protein expressed in P. pastoris (wtAQP0) results in a significant permeability increase of Chl-rich liposomes (p < 0.05 by comparison of proteoliposomes with pure lipid vesicles at each pH using two-way analysis of variance). This result indicates that both native AQP0 and recombinant wtAQPO are functional. The difference in water permeability of native AQP0 and recombinant wtAQP0 at pH 6.5 and 7.5 is small but statistically significant (p < 0.05). By comparison, the difference in water permeability of pure lipid vesicles at pH 6.5 and 7.5 is statistically not significant (p = 0.1). The error bars represent the standard deviation of the measurements. *p < 0.05, **p = 0.1, #p < 0.05 when compared with pure lipid vesicles. (The p-value is a measure of the probability that the permeability values of vesicles measured under two different conditions will be identical.) Adapted with permission from ref 66. Copyright 2017 Cell Press.

know that lipids interact very specifically with AQP0 such that the acyl chain conformation of lipids in contact with AQP0 is determined both by interactions with the protein and with lipids in the surrounding bilayer.69 Tong et al. and Saboe et al. used lipids with similar head groups but markedly different acyl chains. While Tong et al. used lipids with defined acyl chains (for example, POPC), Saboe et al. used egg PC, brain PS, DMPC, and DOPC. It is not a huge leap to speculate that the differences in acyl chains used in the reconstitutions by the two groups might account for the different experimental results. Thus, the consensus of results from multiple groups is that AQP0 and many of its paralogues have a water permeability that is pH dependent and probably mediated by external histidines.

between lipid and protein may alter the conformation and conformational flexibility of the aquaporin protein, and we G


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Figure 9. Effects of external calcium concentration on the osmotic water permeability. (A) Lowering external Ca2+concentration increases the water permeability induced by AQP0 by a factor of 4.1 ± 0.4. (B) Changing external Ca2+ concentration has no effect on the water permeability of AQP1. (C) The increase in water permeability induced by low Ca2+does not depend on expression level. To obtain a wide range of water permeabilities, we injected 5, 10, 50, or 125 ng of AQP0 cRNA. Expression level, as monitored by permeability under standard conditions of pH 7.5, 1.8 mM Ca2+, is shown on the abscissa. The ordinate is the relative permeability defined as (Pexp − PUI)/(Pst‑PUI), where Pexp is the permeability under experimental conditions (e.g., low calcium or defined pH), Pst is the permeability under standard conditions of pH 7.5, 2 mM Ca2+, and PUI is the permeability of uninjected oocytes. Each data point is the average of nine measurements (three different batches of oocytes, three oocytes from each batch). Adapted with permission from ref 63. Copyright 2000 American Society for Biochemistry and Molecular Biology.

Figure 10. Effect of internal calcium concentration on the osmotic water permeability. (A) Injection of BAPTA (2 mM) into the oocyte 30 min before the swelling assay increases AQP0-induced water permeability by a factor of 3.5 ± 0.6 and eliminates sensitivity of water permeability to external Ca2+. LPA (10 μM) added to the bath elevates internal Ca2+ and prevents the increase in AQP0 water permeability normally induced by low external Ca2+. (B) BAPTA has no effect on AQP1. Each data point is the average of nine measurements (three different batches of oocytes, three oocytes from each batch). Adapted with permission from ref 63. Copyright 2000 American Society for Biochemistry and Molecular Biology.

Although we now know for sure that pH can regulate the Pf of AQP0 and that this regulation is mediated by external histidines, we do not know the role pH regulation plays or may play in lens function and development. Because pH regulation is so widespread and variable, especially in fish, it seems a good bet that it does play an important role (or roles) at least under some conditions. Knockout/rescue experiments in which mutants with altered pH regulation are swapped for wild typ AQP0 species may be able to shed light on this question in the near future. Fish seem an especially good place to start because the variety of pH sensitivity among fish AQP0s is greater than that in other species.

found that coinjection of the dominant negative Catfr mouse AQP0-LTR RNA (which lacks water permeability) along with WT AQP0 RNA into Xenopus oocytes resulted in a water permeability that lacked sensitivity to Ca2+, and they suggested that this interaction with WT AQP0 impeded the ability of transgenic expression of WT AQP0 in Catfr mice to support proper lens development and homeostasis.52 This was the first clue, albeit indirect, that calcium regulation of water permeability of AQP0 Pf might be physiologically important and not just a biophysical curiosity. Clemens et al. showed that while morpholino knockdown of AQP0 could be rescued with an expression construct of either WT Aqp0a (from zebrafish) or MIPfun, it could not be rescued by a mutant lacking Ca2+ regulation.54 This finding strongly supported the original contention of Kalman et al. that Ca2+ regulation of AQP0 is a bona f ide developmental necessity for the lens.

3.3. Regulation by Ca2+

Regulation of AQP0 Pf by calcium is another story and has not been studied experimentally as extensively as pH regulation. Calcium regulation of AQP0 has, however, been directly and indirectly shown to influence lens development. Kalman et al. H


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described in Subsection 5.3. These results confirmed that the AQP0−CaM complex has a stoichiometry of four AQP0 monomers (or one tetramer) and two CaM monomers. Each CaM monomer interacts with the C-termini of two AQP0 monomers with the C-termini bound to the CaM molecule as antiparallel dimers, confirming an earlier structural model based on NMR and calorimetric data.71 All-atom MD simulations, described in more detail in Subsection 5.3, showed that CaM modulated the access of water to constriction site II (CS II) near the cytoplasmic side of AQP0 by allosterically restricting the movement of Y149. Thus, the combination of structure and MD simulation identified Y149 as a critical gate regulating the Pf of AQP0.70 Reichow et al. tested this prediction by mutating Y149 to Gly, Leu, and Ser in AQP0 (see Figure 17e in Subsection 5.3 below). As expected, AQP0-Y149G had a high Pf that was insensitive to Ca2+. The AQP0-Y149L Pf was low and also insensitive to Ca2+ concentration. AQP0-Y149S had blunted sensitivity to Ca2+ but still exhibited some change in Pf with low Ca2+. Thus, we understand the mechanism of Ca2+ regulation of Pf at the molecular level in some detail. We also have experimental confirmation that such regulation is essential for a clear lens,54 although we do not yet have a mechanistic notion of why this is so.

Figure 9A shows that lowering external calcium increases AQP0 Pf. Moreover, the same manipulations of 2 mM Ca2+, EGTA, no Ca2+ added, or 10 mM Ca2+ have no effect on the water permeability of AQP1, as shown in Figure 9B.63 Figure 9C shows that the expression level of AQP0 has no effect on the relative change of Pf, ruling out alterations of trafficking as an explanation of the permeability changes. Although the results of Figure 9 show that lowering the external Ca2+ concentration increases Pf, it is actually the internal Ca2+ concentration that effects the permeability change. The first result suggesting that is shown in Figure 10A which compares the effect on Pf of 2 mM external calcium and no external calcium, under control conditions, and when the oocytes expressing AQP0 were injected with 1,2-bis(2aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid (BAPTA), which locks internal calcium low, or with lysophosphatidic acid (LPA), which locks internal calcium high. In BAPTA-injected oocytes, Pf is insensitive to external Ca2+ and locked high. In LPA-injected oocytes, Pf is also insensitive to external Ca2+ but is locked low. Thus, while the external Ca2+ concentration apparently alters the internal Ca2+ near the cytoplasmic mouth of AQP0, it is the internal Ca2+ concentration that effects the change.63 AQP0 does not sense the internal concentration Ca2+ concentration; rather, it senses the concentration of Ca2+bound calmodulin (CaM). This was first demonstrated by Németh-Cahalan et al. using crippled CaM, a mutant that cannot bind Ca2+, and CaM inhibitors.65 Figure 11 shows that

3.4. Cooperativity

An interesting aspect of both pH regulation and Ca2+ regulation of AQP0 Pf is that, even though each monomer in the AQP0 tetramer has its own water pore, these pores are not independently regulated. This is best shown by coinjecting oocytes simultaneously with different RNA ratios of WT AQP0 and mutant AQP0 lacking either pH or Ca2+-sensitive Pf. Cooperativity first manifested itself in the Pf response to Zn2+.72 WT AQP0 responds to 1 mM Zn2+ with an increased Pf but the mutants H122Q and H40C do not respond to Zn2+ (Figure 13A). AQP0 is always tetrameric, and if the monomers insensitive to Zn2+ do not influence the Pf of WT, then the Pf of a mixture of WT and insensitive monomers should just be a linear combination of the Pf values of the two types (as shown by the dashed line in Figure 13C). But, this is not what happens. As Figure 13C shows, adding insensitive monomers reduces the Pf more than expected if the monomers act independently in the tetramer. The solid curve in Figure 13C shows the prediction assuming a single insensitive monomer is sufficient to render the whole tetramer insensitive to Zn2+. Each monomer has its own permeation pathway, but the monomers do not always act independently. For example, a single monomer insensitive to Zn2+ can render the entire monomer insensitive. pH and Ca2+ regulation exhibit similar cooperativity.73 The H40C mutant, which is insensitive to Zn2+, is also insensitive to acidic pH but does increase its Pf in response to basic pH. So, for acid pH, H40C is the insensitive monomer but, for basic pH, WT AQP0 is the insensitive monomer (Figure 14). The green solid line in Figure 14B shows the fit for independent monomers (that is, monomers that do not act cooperatively; here, the insensitive monomer is WT, which does not respond to basic pH). The red dashed line shows the fit for one insensitive monomer rendering the whole tetramer insensitive (here, the insensitive monomer is the mutant H40C).

Figure 11. Elimination of Ca2+ sensitivity of the bAQP0, bAQP0/ H40C, and MIPfun Pf by crippled CaM (a mutant that cannot bind Ca2+). (A) The water permeability of bAQP0 and crippled CaM coinjected oocytes is still increased by low pH but not by low Ca2+. (B) The water permeability of bAQP0/H40C and crippled CaM coinjected oocytes is not increased by low pH or low Ca2+. (C) The water permeability of MIPfun and crippled CaM coinjected oocytes is not increased by low pH or low Ca2+. Note the different scale for MIPfun. Adapted with permission from ref 65. Copyright 2004 American Society for Biochemistry and Molecular Biology.

introducing crippled CaM into the oocyte expressing different AQP0 constructs eliminates Ca2+ sensitivity of the water permeability but does not affect the sensitivity of Pf to pH for either AQP0 or MIPfun. In a more detailed study of the interaction between AQP0 and CaM, Reichow et al. provided a structural and functional basis for the CaM modulation of AQP0 Pf.70 They reported a medium-resolution EM structure of the AQP0−CaM complex (Figure 12) and a supporting MD simulation that will be I


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Figure 12. Purification and pseudoatomic model of the AQP0−CaM complex determined by EM. (a) Chromatogram showing purification of the AQP0−CaM cross-linked complex from excess free CaM by size-exclusion chromatography. mAU, milliabsorbance units. (b) Silver-stained SDSPAGE showing fractions from the size-exclusion chromatography purification. Lane 1, total starting material; lane 2, purified AQP0−CaM crosslinked complex, dissociated in SDS into three protein bands migrating at 26, 39, and 65 kDa, corresponding to the AQP0 monomer, the 1:1 AQP0−CaM cross-link, and the 2:1 (AQP0)2−CaM cross-link, respectively; lane 3, free CaM migrating as a diffuse band at ∼13 kDa. (c) Electron micrograph of negatively stained AQP0−CaM particles. Inset, symmetrized projection averages of the AQP0−CaM complex. (d) Different views of the 3D reconstruction of the AQP0−CaM complex. (e) Fitting of the crystallographic structures of AQP0 (orange and yellow) and CaM (blue) into the 3D reconstruction (gray mesh). (f) Pseudoatomic model of the AQP0−CaM complex displaying two CaM molecules (A and B) bound to the cytoplasmic C-terminal helices of the AQP0 tetramer. Reproduced with permission from ref 70. Copyright 2013 Nature Publishing Group.

The increase in Pf induced by Ca2+ has a similar pattern of one stimulus eliciting a cooperative response and one stimulus producing an independent response of each monomer type. In the case of Ca 2+ , WT AQP0 responds to low Ca 2+ concentration by an increase in Pf but does not respond to an increase in Ca2+ from 2 to 5 mM. The mutant S235D does not respond to low Ca2+, but 5 mM Ca2+ increases its Pf about 2-fold. In mixtures of WT and S235D, WT can suppress the Pf increase induced by 5 M Ca2+, but S235D and WT act independently in responding to low Ca2+.73 Thus, the interaction between monomers in an AQP0 tetramer can be critical in determining the response of the tetramer as a whole to a given stimulus. For the acid pH

stimulus, a single insensitive monomer renders the whole tetramer insensitive. The same is true for elevating the Ca2+ level to 5 mM. But, mutants sensitive to basic pH and WT monomers act independently even when in a mixed tetramer. WT monomers and monomers insensitive to low Ca2+ also act independently in a mixed tetramer.

4. MOLECULAR DYNAMICS SIMULATIONS OF AQPs 4.1. Molecular Dynamics Simulations of Membranes and Membrane Proteins: A Brief History

Around 1990, the field of membrane simulations naturally sprung out as an offshoot from the field of biopolymer J


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Figure 13. (A) Effect of 1 mM ZnCl2 on the osmotic water permeability of coinjected AQP0 and a mutant insensitive to zinc H122Q or H40C. Mix 1:1 represents 10 ng of wild type and 10 ng of mutant H122Q (fraction of insensitive monomer = 10 divided by [10 + 10] = 0.5) and mix 5:1 represents 10 ng of wild type and 2 ng of mutant H122Q (fraction of insensitive monomer = 2 divided by [10 + 2] = 0.166). In both mixtures, the effect of zinc was dramatically diminished, but the calcium sensitivity remained intact. Mix 1:1 represents 5 ng of wild type and 5 ng of mutant H40C. (B) Western blot of uninjected oocyte membranes, AQP0, mutant H122Q, and mutant H40C. A band, lower than the 28 kDa marker, was seen in AQP0, H122Q, and H40C and not seen in uninjected oocytes. The bar graphs represent the average of scanned bands from three different experiments. There was no noticeable difference between the wild type and the mutant expression level. (C) Theoretical curves predicting the factor of increase, assuming the monomers behave independently (gray dashed line) or one or two insensitive monomers can block zinc sensitivity (black continuous line and gray dotted line respectively). Experimental results are plotted as filled squares and are well-fit by the one insensitive monomer curve. Each data point is the average of experiments using nine oocytes from three different batches. Reproduced with permission from ref 72. Copyright 2007 Rockefeller University Press.

membranes. The first was that membranes are essentially twodimensional, semi-infinite systems, so that the minimal system size required to model a patch of membrane using periodic boundary conditions without introducing artifacts was relatively large. The second was that the delicate balance between the weak interactions in the nonpolar membrane interior and the strong interactions in the polar headgroup region and membrane-water interface presented new challenges to force field development. The third was that the symmetry of membranes prompted the need for more accurate and computationally demanding treatments of long-ranged electrostatic forces (e.g., Ewald lattice sums) to replace the inadequate spherical truncation approach that was typical of biomolecular simulations of the day. Finally, efficient sampling of even the most elementary motions in membranes, torsional transitions in the acyl chains, required relatively long simulations. The simulation often cited as the first atomistic MD simulation of a complete lipid bilayer, including polar

(primarily protein, but also nucleic acid) simulations, which by then had been maturing for about 15 years. The vast majority of biomolecular simulations are atomistic MD simulations that consist of the numerical integration of classical equations of motion driven by forces computed from an empirical potential energy function, or “force field.” MD trajectories are thereby determined over a sequence of elementary time steps, which for the most commonly employed atomistic models is roughly one fs. The first MD simulation of a soluble protein generated a trajectory of less than 10 ps duration (a few thousand time steps) for the bovine pancreatic trypsin inhibitor, a small protein containing only 58 amino acids, in vacuum, on a mainframe computer.74 Despite its short duration and the lack of solvent, this simulation demonstrated the validity and utility of molecular mechanics force fields for modeling biomolecules, and showed that proteins are remarkably flexible, with a “fluidlike” interior, even on a timescale of ∼10 ps. The relatively late advent of membrane simulations was a consequence of the unique technical challenges presented by K


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protocols for phospholipid bilayer simulations. A wave of publications (reviewed by Pastor76 and Tobias et al.77) reporting the initial efforts of the pioneering groups appeared in the early to mid-1990s, a period that could be regarded as the “formative years” of membrane simulations. The early efforts of the Berendsen group75,78,79 defined and scrutinized the methodology used to simulate membranes. The highly visible 1993 Science paper by Pastor and coworkers, comparing the fluidity of the interior of a lipid membrane to a long-chain alkane liquid, demonstrated the potential of MD simulations for providing important new insights into membrane physical properties and lipid dynamics in membranes.80 Stouch reported one of the most extensive analyses of the first simulation of a phospholipid bilayer carried out for longer than 1 ns.81 The Klein group introduced several simulation techniques that were initially considered radical in the biomolecular simulations community but are now standard and almost universally employed in membrane simulations. These include Ewald summation of electrostatic interactions, performing membrane simulations in the isothermal−isobaric (NPT) ensemble (with an isotropic pressure tensor corresponding to vanishing membrane surface tension) and multiple time step integration of the equations of motion.82−84 The Klein group also reported the first nanosecond timescale simulations of phospholipid bilayers at constant temperature and pressure.82,83 Berkowitz and coworkers performed some of the earliest simulations of hydrated phospholipid bilayers to carry out a detailed analysis of water properties near membrane surfaces.85,86 During this period, Berkowitz, Darden, and coworkers introduced an efficient version of the Ewald sum referred to as “particle mesh Ewald” (PME),87 which has had an enormous impact on biomolecular simulations. In the late 1990s, the Ewald sum started to become the standard approach to treating electrostatic interactions in membrane simulations, thanks in large part to PME. Merz and coworkers reported some of the earliest atomistic MD simulations of both PC and PE bilayers,88−90 as well as one of the first simulations of a peptide interacting with the surface of a membrane.91 The first MD simulation of a transmembrane peptide (gramicidin A) was reported by Woolf and Roux92 in 1994. This pioneering work paved the way for the modeling of membrane-embedded ion channels, which represents a significant fraction of the total number of applications of membrane simulations carried out to date. Huang and Loew93 performed the first MD simulation of an amphipathic helix (residues 13−41 of corticotrophin-releasing factor) embedded in a bilayer-water interface. The simulation of bacteriorhodopsin (bR) in a model of its native purple membrane by Edholm et al., reported in 1995, was the first simulation of a bona f ide multipass membrane-embedded protein (containing seven transmembrane helices).94 This pioneering simulation was made possible by the existence of a high-resolution structure,95 the second of a membrane protein. The vast majority of MD simulations of membrane proteins use experimentally determined high-resolution structures as input. By the end of 1993, the year when the first MD simulations of phospholipid bilayers were reported, there were only five unique membrane protein structures in the Protein Data Bank (PDB).96 During the period when MD simulations of membranes were coming of age, the pace of membrane protein structure determination began to pick up, so that, by the end of 2000, the year that the first structures of the water channels AQP132 and GlpF39 appeared in the literature, there

Figure 14. Cooperativity in the pH regulation of AQP0. (A) Effect of acid and alkaline pH on the water permeability of mixtures of AQP0 (WT) and the H40C mutant. WT responds to acid pH, whereas the H40C mutant responds to alkaline pH. Mix 5:1 represents 10 ng of AQP0 and 2 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.166), and mix 1:1 represents 10 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 10/20 = 0.5). The Pf of the mix 1:1 at pH 8.5 is greater than the Pf at pH 7.5 with a pvalue of 5 × 10−5 (Student’s t test). The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. (For acid pH, the H40C mutant is the insensitive monomer. For alkaline pH, WT is the insensitive monomer.) Experimental results for acid pH are plotted as red squares and are well-fit by a curve calculated from the binomial distribution of monomers randomly partitioning into each tetramer and the assumption that a single insensitive monomer renders the whole tetramer insensitive to acid pH (dashed red line; χ2 = 0.840). The curve assuming that two insensitive monomers are required to render the tetramer insensitive to acid pH (dashed black line; χ2 = 8.88) does not fit the experimental data. Experimental results for alkaline pH are plotted as green circles and are well-fit by a straight line (the theoretical prediction assuming each monomer acts independently of the others in the tetramer; χ2/DOF = 0.071 and R2 = 0.910; solid green line). Each data point is the average of experiments using nine oocytes from three different batches. Reproduced with permission from ref 73. Copyright 2013 Rockefeller University Press.

headgroups, water, and counterions, was reported by Egberts and Berendsen in 1988.75 Although it was actually a simulation of the lamellar phase of a ternary alcohol-fatty acid−water mixture, it introduced methodological protocols and analysis techniques that would be widely applied in subsequent simulation studies of more biologically relevant lipid bilayers. It was also noteworthy because it demonstrated that a stable, albeit highly disordered, bilayer-water interface could be maintained without restraints over the ∼100 ps timescale of the simulation. Diacylphospholipids are the most abundant lipids in many biological membranes. In the early 1990s, several groups were working on developing the force fields and simulation L


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were more than 40 unique membrane protein structures.96 Simulators were hungry for membrane protein structures, and it seemed that each report of a new structure was quickly followed by one or more simulation studies based on that structure. AQPs were no exception.

NH2 groups in the side chains of the asparagine residues in the NPA motifs and the backbone atoms in the two half helices that meet at the NPA motifs led to a uniform orientational distribution of water dipoles in the channel, thus confirming the role of the helix dipoles in the net alignment of water molecules in the two halves of the pore. Based on pore water densities in their crystal structure and MD simulation, Tajkhorshid et al. hypothesized that obstruction of the pore at the ar/R constriction site was the cause of the slow water conduction of GlpF relative to AQP1, according to the available experimental data at the time.41 To test this hypothesis, they considered a double mutant, W48F/ F200T, aimed at reducing the size of the constriction and increasing the polarity of the ar/R region. Both light scattering assays and MD simulation of the double mutant indicated increased water permeability vs WT GlpF, and an X-ray crystal structure and the MD simulation of the double mutant exhibited increased water density in the ar/R constriction region. Thus, the combination of experiments and MD simulations of the W48F/F200T double mutant confirmed the crucial role of the ar/R “selectivity filter” in limiting the rate of water conduction in GlpF. Both of the MD simulation studies reviewed in the last three paragraphs attributed the inability of AQPs to conduct protons, in part, to disruption in the continuity of hydrogenbonded chains of water molecules.40,41 However, these simulations did not include protonated water species, and the mechanism of proton exclusion had to be revised after protons were explicitly included in subsequent simulations. For a detailed account of the evolution of thinking concerning proton exclusion, the reader is referred to the review by de Groot and Grubmüller.114 The consensus picture that emerged from seven studies that were reported in 2003 and 2004 is that, while protons can be efficiently transferred via Grotthuss hopping throughout the length of the water-conducting pore,101 a high free energy barrier dominated by electrostatic interactions prevents the transfer of protons across the NPA motifs.101−103,105,107,118,119 The electrostatic barrier was robust in the sense that it consistently emerged from multiple research groups employing a diverse panel of computational approaches, and its origin could be traced to the two half helices (e.g., HB and HE in AQP1 or M3 and M7 in GlpF) that meet at the NPA motifs. The macrodipoles of the two half helices are oriented such that there is a positive electrostatic potential, which is repulsive to positively charged proton species, at the NPA motifs. The role of the half helices was clearly demonstrated by a simulation study of GlpF that showed that the barrier to proton conduction was greatly reduced when the charges on the NPA motifs and the backbone atoms of the half helices were set to zero.107

4.2. Mechanistic Details of AQP Water Conduction, Gating, and Selectivity Revealed by MD Simulation Studies

Within a year or so of the publication of the first AQP1 and GlpF structures, four pioneering MD simulation studies based on those structures were published.40,97−99 Within five years, 18 computational studies of these AQPs had been reported.40,41,97−112 The majority of these studies were aimed at elucidating the generic mechanisms of AQP water conduction, gating, and selectivity. Although this work has been reviewed previously (see, e.g., reviews by Fujiyoshi et al.,113 de Groot and Grubmüller,114 Hedfalk et al.,115 Fu and Lu,116 and Hub et al.117), we provide a brief summary in this Subsection because some of the mechanistic details revealed by early MD simulations of AQP1 and GlpF are shared by AQP0, which is the main subject of the present review. As we shall see in subsequent Subsections, understanding the relatively low permeability of AQP0 compared to other AQPs requires additional mechanistic insights that have also been provided by MD simulations. In 2001, the first four atomistic MD simulation studies of AQPs (AQP1 and GlpF) were reported.40,97−99 In only one of the studies were the simulations of AQP1 and GlpF sufficiently long (10 ns) to observe full water permeation events and, hence, provide genuine insights into the water conduction mechanism.40 Based on their observations, de Groot and Grubmüller described both proteins as “two-stage filters”, with the first stage at the NPA motifs and the second in the ar/R constriction region. Protein−water hydrogen-bond energies were lowest (most negative and, hence, more favorable) and compensated for the loss of water−water hydrogen bonds, thus lowering the free energy barriers to water passage in both stages. In the hydrophobic regions adjacent to the NPA motifs, such compensation is not possible, so the rate-limiting barriers to conduction in the free energy profile for water molecules in the pore were located in the hydrophobic regions. The disruption of hydrogen-bonded water chains was observed to be greatest in the ar/R region. This observation, along with the presence of the positively charged arginine side chain, led de Groot and Grubmüller to suggest that the ar/R region is the selectivity filter that blocks the passage of protons and other cations. These authors also observed that, as originally proposed by Murata et al. based on their structure of AQP1,32 the water dipole moments were aligned in one direction in one half of the channel, and the dipoles in the other half were aligned in the other direction, with the center of inversion located at the NPA motifs. The alignment pattern was attributed to the electric fields arising from the macrodipoles of the two half helices HB and HE (Figure 2). A similar alignment of water molecules was also observed by Tajkhorshid et al. in their 12 ns MD simulation of GlpF, during which they also observed full water permeation events.41 The authors argued that this “global orientational” tuning would lead to proton exclusion in two ways. First, water molecules are unfavorably aligned at the ends of the pore for proton uptake and release and, second, the continuous hydrogen-bond chain required for conduction via the Grotthuss hopping mechanism is broken at the NPA motifs. Turning off the charges of the

4.3. Making Contact with Experiment: Calculating pf from MD Simulations

For structural and mechanistic insights from atomistic MD simulations of complex biomolecular systems such as membrane-embedded proteins to be trusted, the MD simulations must first be validated against experimental data. The ideal test is a side-by-side, quantitative comparison of an experimental measurement of a key observable against the corresponding computation of the same quantity from an MD trajectory generated under the same conditions as the experiment. The natural target for atomistic MD simulations of AQPs is the single-channel osmotic water permeability M


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Figure 15. Atomistic model and the curve of water flow through AQP3 induced by an osmotic gradient at 22 °C. The atomistic model system illustrated in the top panels consists of four tetramer complexes of AQP3 (16 monomer channels) embedded in a 218 Å by 222 Å patch of PE (16:0/18:1) lipid bilayer. On each side of the membrane there is a 40 Å-thick layer of saline. One side has 150 mM NaCl, while the other has 545 mM NaCl. The two sides of the membranes were prevented from artificial mixing as in the usual periodic boundary condition.122 Water can only exchange between the two sides via permeation through AQP3 channels. The 16 protein monomers are shown as ribbons so colored that each channel has its unique color. Salt ions are shown as large cyan and gold spheres. Waters and lipids are shown in lines colored by atom names (H, white; C, cyan; O, red; N, blue; P, purple). All molecular graphics in this figure were rendered with VMD.42 Reproduced with permission from ref 123. Copyright 2019 Elsevier.

(similar to the swelling assays reported in43) would generate the net transport of only ∼6 water molecules per 1 μs of trajectory. This low number of permeation events must be contrasted with the effects of thermal fluctuations, which can be estimated from measurements of the diffusional water permeability.120 The reported ratio of osmotic permeability to diffusional permeability for AQP1125 suggests that thermal fluctuations would produce permeation events at a rate of ∼120 waters/μs. This implies that the measured osmotic water flow from a simulation of a single biological unit under an osmotic gradient similar to experiment will likely be too noisy to be reliably compared to experiments, at least for pf values on the order of 10−14 cm3/s or less. Alternative approaches explored in the early literature were to increase the magnitude of the magnitude of the explicit osmotic gradient121 or the corresponding osmotic pressure difference.100,104 The first simulation of osmotically driven water flow was performed using carbon nanotubes as model

coefficient, pf, which can be estimated from osmotically driven water flow experiments in whole cells (e.g., oocytes) or vesicles by measuring the corresponding Pf values and counting the number of expressed channels. Under the usual experimental conditions of these assays, pf can be expressed as120 pf =

jw Δosm

where jw the net water flow rate through the channel (in mol/ s) and Δosm is the osmotic gradient (in mol/cm3). In principle, it is possible to mimic the experimental situation with a nonequilibrium MD simulation under an osmotic gradient by setting up two separate solution compartments with different salt concentrations.121−124 To assess the feasibility of such an approach, let us consider the case of AQP1, for which the experimental value of pf, measured in oocytes, is 1.2 × 10−14 cm3/s.43 Thus, a simulation of a single AQP1 tetramer under a ∼200 mM osmotic gradient N


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pores under an explicit ∼6 M osmotic gradient.121 This simulation confirmed that water transport through nanopores is governed by thermal fluctuations and consequently cannot be modeled as continuous (e.g., Poisseuille) fluid flow.121 Zhu et al. developed a single solvent compartment methodology whereby explicit forces along the transmembrane direction were applied to water molecules in the central region of the solvent, amounting to a hydrostatic pressure difference across the membrane of 1000−2000 bar.104 A 5 ns simulation of AQP1 produced measurable net water flow that was consistent with experimental data and confirmed, at least qualitatively, the adequacy of the prevailing model of single-file water transport.104 However, the imposition of such a large pressure difference over a limited trajectory length required the use of harmonic constraints to stabilize the protein structure, thereby limiting the extent of the mechanistic insights that could be drawn from the simulation. The first ∼10 ns timescale equilibrium MD simulations of AQP1 and GIpF permitted the direct counting of a sufficient number of permeation events facilitated by thermal fluctuations to produce permeation rates that were consistent with experimental data.40,41 Based on these results as well as similar results from simulation of water permeation through carbon nanotubes126 and gramicidin A,127 a stochastic description of water flow through narrow pores arose that made it possible to obtain estimates of pf using equilibrium MD simulations.104,127,128 The stochastic model relies on the assumption that, in a water-filled narrow pore where waters may, ideally, form a single file, water molecules move in a concerted manner in discrete steps (hops) on the order of the diameter of a water molecule. In the absence of an osmotic gradient, these hops would occur with similar rate in both directions at a rate, k0, proportional to pf, i.e.,104,127

Nonequilibrium MD simulations under an explicit osmotic gradient have been revisited recently by Chen and coworkers.122,123 In their simulation scheme, a single membrane system containing four AQP tetramers is placed between two separate solution compartments (Figure 15). Their studies have been focused on fast-conducting AQP3, AQP4, AQP5, and GlpF channels, which allows the use of moderate osmotic gradients ( AQP1 ≫ AQP0, which is in rough agreement with the ranking of quantitative experimental pf measurements: GlpF > AQP1 > AQPZ ≫ AQP0 (see a recent review of water permeabilities of membrane channels and transporters,129 which compiles pf values of AQP0, AQP1, AQPZ, and GlpF). The authors measured the relative pore sizes, obtaining the ranking GlpF ≫ AQP1 > AQPZ ≫ AQP0, which indicates some, but not complete, correlation with the experimental pf values. Based on a water displacement cross-correlation function, they identified the compressibility or single-file character of the water in the pores as an additional factor determining pf. Single-file character decreases with increasing pore size so that the permeation rate does not increase with pore size. The low pf of AQP0 was attributed to the lack of a single file of water molecules in the narrow pores as well as the presence of the pore-blocking Y149 side chain at the CS II. In a follow-up to their pioneering study comparing AQP0 to other AQPs, Hashido et al.130 reported a new analysis of their previous simulations of AQP0, AQP1, AQPZ, and GlpF along with new simulations (also 5 ns duration in POPC bilayers) of AQP4; three mutants of AQPZ, L170 V (in the NPA region), N182G (outside the ar/R region), and the L170 V/N182G double mutant, meant to mimic AQP1; and a carbon nanotube. The objective of the new analysis methodology

pf = vwk 0

where vw is the average volume of a single water molecule. Transition state theory can be invoked to justify this relationship by comparing experimental measurements of pf against experimentally determined water transport activation energies.129 An approach to estimating k0 from equilibrium MD simulations was developed by Zhu et al.,110 whereby the net number of hops as a function of time are measured by a collective coordinate, n(t), defined by accumulating all of the instantaneous fractional displacements of water molecules within the length of the pore. In this manner, the net translocation of one water molecule through the channel will change n(t) by ±1. At equilibrium, n(t) is described by a simple random walk with ⟨n(t)⟩ = 0 and ⟨n2(t )⟩ = 2k 0t

As stressed by Zhu et al., the strength of this description is derived from the fact that, by using instantaneous displacements along the transmembrane direction instead of individual hops, it applies to cases such as aquaporins that do not conform to the ideal quasi one-dimensional single-file channel configuration.110 This methodology has been applied successfully to several simulations of aquaporin channels on the submicrosecond timescale, including AQP1 and AQP0,66,130−132 but it did not provide an adequate pf estimate for the faster GlpF channel.130,133 O


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Additional simulations in which the WT AQP0 and the Y23F mutant were restrained in either the open or closed end states of the FMA modes showed that the pf of WT AQP0 was as high that of the Y23F mutant only when the R187 gate was held open. Thus, this study put the spotlight on R187 in the ar/R region as a key gating residue in AQP0.

was to provide a direct connection between the experimentally measurable single value of pf and the pore structure. To this end, they extended the collective diffusion model of Zhu et al.104 by dividing the pore lumen into subchannels so that the single value of pf becomes a so-called osmotic permeability matrix with elements pij; the diagonal elements give the permeabilities of the subchannels, and the off-diagonal elements the covariances between water molecule displacements in two different subchannels. The analysis revealed that, in all of the AQPs considered, correlated motion of water molecules was reduced in the NPA region, although the degree of the reduction varied among the AQPs. For example, AQPZ, which exhibited single-file-like permeation, had the strongest correlation; while GlpF, with its large pore, exhibited a relatively low correlation, and AQP0 exhibited almost no correlation across the NPA region due to occlusion by Y23. Although the local permeability in the ar/R region is low in all of the AQPs due to strong protein−water interactions, the analysis did not reveal a significant reduction of correlated water motion in that region. Finally, the analysis of the three AQPZ mutants showed that the lower pf of AQP1 relative to AQPZ predicted by the simulations is due to broadening of the pores in the NPA and outside of the ar/R regions. The key role for Y23 in AQP0 gating was corroborated in a simulation study by Qiu et al.,132 who carried out 60 ns MD simulations in POPE bilayers of AQP1, AQP0, and the Y23F variant of AQP0, which mimics AQP1 because the residue corresponding to Y23 in AQP1 is F24. Using the collective diffusion model,104 the authors calculated pf values for AQP0 and AQP1 that were in good agreement with experimental measurements, and their AQP0 pf was ∼10 times lower than their AQP1 pf. The pf of the Y23F variant of AQP0 was ∼4 times higher than that of wild-type AQP0, confirming a role for Y23 in the slow water conduction of AQP0. A comparison of water density profiles in the pores revealed that water was significantly depleted in AQP0 relative to AQP1 near the locations of Y23 and Y149 in AQP0; the corresponding free energy profiles (also known as potentials of mean force or PMFs) displayed a free energy barrier of ∼4.5 kcal/mol at Y23 in AQP0, which is substantially higher than the highest barrier (∼1.5 kcal/mol) in the AQP1 pores. The roles of Y23 and Y149 in determining the slow water conduction of AQP0 were the focus of a joint experimental and computational study reported by Saboe et al.66 The Pf values measured experimentally in AQP0 proteoliposomes for the Y149T and Y23F mutants, in which AQP0 residues are replaced with the corresponding residues in AQP1, were roughly two times and 20 times higher, respectively, than the Pf of WT AQP0, and effects of the two mutations on Pf were additive in the Y23F/Y149T double mutant. The trend in the experimental Pf values for WT AQP0 and the two single mutants was reproduced qualitatively in the single-channel pf values computed from MD simulations of several hundred ns duration. However, the pf computed for the double mutant was similar to that of the Y23F mutant, i.e., the simulation did not reproduce the additivity of the two mutations. The authors attributed this discrepancy to the incomplete relaxation of the structure of the protein following the large perturbation resulting from the double mutation. Processing of the MD trajectories using the so-called functional mode analysis (FMA)134,135 revealed three modes, dominated by motions of R187, Y23, and Y149, respectively, that were correlated with the increases in pore radii that accompanied the mutations.

5.2. Simulation Studies Comparing Junctional and Nonjunctional AQP0

In addition to serving as a water channel, AQP0 also has an adhesive role, forming thin junctions between lens fiber cells consisting of AQP0 square arrays.136−138 Prior to the simulation studies summarized in this Subsection, the water permeability of the junctional form of AQP0 was under debate, and the biological significance of the low pf of AQP0 was uncertain. The simulation studies summarized in this Subsection sheds light on both of these issues. Han et al. reported out the first comparative MD simulation study of AQP0 in both the junctional form in a double lipid bilayer configuration and the nonjunctional form in a single bilayer.139 They included AQP1 in their study to gain insights into the structural and dynamical differences that lead to the much lower pf in AQP0 vs AQP1; the trajectory lengths were 1.3 ns for AQP1 and 10 ns for AQP0. We first summarize the results of the single-bilayer systems. By directly counting the number of water molecules that completely transited the pore per unit time, the authors estimated that, in decent agreement with experimental measurements, the water permeation rate of AQP1 is about 16 times greater than that of nonjunctional AQP0. The authors observed that, in their simulation of the EM structure, the extracellular constriction residues R187 and H172, which appear to block the pore in the EM structure, move into positions and orientations similar to those found in the X-ray structures of AQP0 and AQP1 in most pores but also exhibit different orientations in some pores. It was also noted that the remaining two constriction zones in AQP0 at Y23 and Y149 opened and closed through random movements of the tyrosine side chains and that the open configuration of Y23 was substantially less probable than that of Y149. The time evolution of distances between key residue pairs in the constriction zones indicated that the pores were substantially wider and more stable in AQP1 than in AQP0. As expected, the PMF for water translocation across AQP0 exhibited substantially higher free energy barriers than for AQP1: the mean barrier heights were found to be 1.74 kcal/mol for AQP0 and 0.42 kcal/mol for AQP1. Despite the presence of an extended tetramer−tetramer interface in the junctional form of AQP0 in the double bilayer simulation setup, water was still able to transit the full length of the AQP0 pores, albeit at a rate that was half that of the single-bilayer configuration. The authors attributed the lower permeability to a restriction of the motion of the helices due to loop region contacts between the opposing tetramers. Taking advantage of recent advances in algorithms for MD simulations on parallel computers, Jensen et al.131 were able to carry out MD simulations of both the nonjunctional (tetrameric) and junctional (octameric) forms of AQP0 on the 100 ns timescale, i.e., an order of magnitude longer than prior simulations of AQP0. Jensen et al. showed that obtaining converged pf values using the collective diffusion model104 for AQP0 required ∼100 ns length trajectories. Consistent with Han et al.,139 Jensen et al. found the junctional form of AQP0 to be conductive, but they predicted essentially the same pf for P


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Figure 16. Luminal residues modulate transport. Snapshots from the simulation of the single AQP0 tetramer initiated from the structure of the junctional form that represent open and closed states of AQP0. The configurations of Arg-187 and Tyr-149 are classified as “open” or “closed” depending on whether they block water passage and as “up” or “down” depending on their side-chain dihedral angles. A time-averaged water density profile ρ(z), drawn to scale with the AQP0 lumen, is overlaid in A; dashed lines indicate standard deviations among 13 separate 12 ns windows. A free energy profile, G(z) = −RT ln[ρ(z))], is overlaid in B. Adapted with permission from ref 131. Copyright 2008 National Academy of Sciences, United States.

model of the AQP0−CaM complex, based on NMR and calorimetric data,71 opened the door for MD simulations aimed at obtaining mechanistic insights into Ca2+ regulation. The first MD simulation of the AQP0−CaM complex actually employed a revised model of the complex, depicted in Figure 12f, that was experimentally validated by cryo-EM and a combination of mutagenesis and isothermal titration calorimetry measurements.70 In both models, CaM binds to the AQP0 CaM-binding domains with a noncanonical two CaM to one AQP0 tetramer stoichiometry, in an orientation that, as Reichow and Gonen first noted,71 appears to block the pores of two AQP0 monomers. Such a blockage would explain why the water permeability of AQP0 is reduced by roughly a factor of 2 in the presence of a physiological concentration of intracellular Ca2+ (Subsection 3.3 and Figure 17e). The first MD simulation of the AQP0−CaM complex showed that CaM binding to AQP0 does not sterically impede water flow through the lumen but rather influences the CS II through an allosteric effect.70 Depending on the orientation of the side chain of Y149, the cytoplasmic gate at the CS II is either open or closed (Figures 16 and 17b−d). In the absence of bound CaM, this gate is mostly open, but when CaM binds, the atomic fluctuations of the AQP0 in the vicinity of the CS II are reduced and the gating equilibrium shifts toward mostly closed states (Figure 17d). As is discussed in Subsection 3.3, oocyte permeability measurements on Y149 mutants (Figure 17e) confirmed the key role of Y149 predicted by the MD simulations in the regulation of AQP0 by Ca2+. Fields et al. used the experimentally validated model of the AQP0−CaM complex reported by Reichow et al.70 to further explore the roles of Ca2+ and phosphorylation in the regulation of AQP0 water permeability.140 Brownian dynamics simulations of the diffusive encounter between CaM and AQP0 revealed that CaM binding is dominated by electrostatic interactions with the cytoplasmic face of AQP0, yet phosphorylation of the AQP0 C-terminal domain does not

the junctional and nonjunctional forms as well as a tetrameric construct of the truncated form that forms junctions. In agreement with previous simulation studies of AQP0, the authors found that Y23 and Y149 are the primary gates to water transport through AQP0, with Y23 acting as a static gate and Y149 as a dynamic gate that swings in and out of the lumen on a timescale of ∼20 ns. The behavior of Y149 is depicted in Figure 16. Jensen et al. observed that R187, which is part of the ar/R or CS I constriction, made transitions between an “up” conformation, in which the pore is open at CS I, and a “down” conformation, in which the pore is blocked at CS I (Figure 16); because R187 was mostly in the “up” conformation, Jensen et al. concluded that it is not responsible for the low pf of AQP0 vs the fast-conducting AQPs. The length of the simulations was sufficiently long to sample a ∼100 ns timescale movement of loop A (Figure 2) that could be relevant for AQP0 regulation by pH (see Subsection 5.3 below). Finally, Jensen et al. presented estimates of the pressure difference across the intermembrane cavity formed between the opposing tetramers in the junction to provide a connection between the low AQP0 pf and the stability of the junction, which is held together by weak hydrophobic contacts. Using AQP0 lumen and cavity pf values as inputs, they estimated that the pressure difference across the cavity is less than 2% of the pressure difference across the entire junction, and speculated that the “low permeability of AQP0 may have evolved to allow it to form stable junctions by decreasing the risk of junction breakage as water flows through.” This intriguing possibility could constitute the missing link between the water channel and adhesive functions of AQP0. 5.3. Simulation Studies of AQP0 Permeability Regulation by Ca2+ and pH

The experimental work reviewed in Subsection 3.3 showed that AQP0 water permeability is regulated by intracellular Ca2+ and established that the Ca2+ regulation is mediated by the calcium-binding protein, CaM. The availability of an atomistic Q


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Figure 17. (a, b) Structures illustrating pore profile analysis of AQP0 during the molecular dynamics simulation. Tyr149 is shown in a downward, open conformation (a; b, left) and in an upward, closed conformation (b, right). The location of CS II is indicated in a. Side chains of the CS II residues Tyr149, Phe75, and His66 are displayed as sticks. (c) Plot indicating the pore diameter of the open AQP0 CSII (blue) and the closed CS II (red). (d) Plot indicating the population of structures obtained during the CaM-free AQP0 (blue) and CaM-bound AQP0 (red) molecular dynamics simulations, clustered according to the displacement of their CS II residues Tyr149 and Phe75. Inset, plot showing the population of structures with CS II displacement 8 Å. (e) Water channel permeability rates (Pf; μm s−1) obtained from oocytes expressing wild-type AQP0 and CS II mutants (Y149G, Y149L, and Y149S) obtained under buffer conditions of 0 mM (blue) or 1.8 mM Ca2+ (yellow). The AQP0 Pf is significantly inhibited by Ca2+ (P = 0.012 by two-sided t test (n = 10 biological replicates)). Inset, anti-AQP0 immunoblot verifying that each construct was expressed and correctly trafficked to the plasma membrane. Reproduced with permission from ref 70. Copyright 2013 Nature Publishing Group.

significantly inhibit CaM binding. Using all-atom MD simulations, Fields et al. discovered that Ca2+ regulation of AQP0 water permeation occurs via CaM coupling to the AQP0 second constriction site (CS II) via a network of interactions through an arginine-rich cytosolic loop of AQP0 located near the center of the tetramer, which includes R153 and R156. Interactions mediated by R156 are depicted in Figure 18. The MD simulations further showed that phosphorylation modulates Ca2+-sensitivity of AQP0 by inducing changes in salt bridging interactions at the CaM− AQP0 interaction interface, thereby modifying the allosteric coupling of the CaM-binding domain to the gating side chain of Y149 at the CS II. Changes in oocyte water permeability upon mutation of R153 and R156 confirmed the simulationderived mechanistic insights into the role of the previously unexplored arginine-rich loop in the regulation of AQP0 permeability.140

Using a special-purpose computer for MD simulations of biomolecules,141 Freites et al. performed multi-microsecond timescale nonequilibrium MD simulations of AQP1, AQP0, and the AQP0−CaM complex under an explicit ∼1.7 M osmotic gradient, and reported good agreement between single-channel pf ratios and the corresponding Pf ratios from oocyte swelling assays.124 The extended simulation timescale allowed the direct characterization of the dynamics of water single-files and their interactions with the amino acid side chains comprising CS-I and CS-II as well as Y23. This analysis led to the identification of open- and closed-state configurations of the single-channel permeation pathway, and an account of the differences in water permeation rates between the systems in terms of changes in the exchange rates between these closed and open state configurations. The allosteric effect upon CaM binding was shown to extend not only to CS-II, as previously reported,70 but also to CS-I, favoring the closing of the channel at both sites. This action was associated with a R


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Figure 18. A chain of electrostatic interactions mechanically couples CaM to the pore-gating residues of AQP0. Snapshots from an MD simulation of CaM-bound WT AQP0. An interaction chain from D118 of CaM to R156 of AQP0 to E151 of an adjacent AQP0 subunit (highlighted by the red enclosure in the first panel) connects CaM to the gating Y149 residue of the CSII. Movement of the Y149 side chain in the third panel follows the breaking of the salt bridge between R156 and E151 in the second panel, resulting in the reformation of the single file of water molecules through cytosolic portion of the lumen. Reproduced with permission from ref 140. Copyright 2017 American Society for Biochemistry and Molecular Biology.

cholesterol to phospholipids in the lens cortex is 1 to 2, while in the lens nucleus it is 3 to 4.144,145 As was mentioned above, lipids were resolved in the high-resolution structures of AQP0 determined by electron crystallography.38,146 In the 1.9 Å structure solved by Gonen et al.,38 a ring of so-called annular lipids was revealed, and annular E. coli polar lipids were found in the 2.5 Å structure determined by Hite et al.146 While these structures provided a detailed yet rare glimpse at membraneprotein lipid interactions, they also raised some questions,147 such as are the positions of the lipids in the two-dimensional crystals representative of the preferred locations of lipids surrounding AQP0 in a fluid lipid bilayer? Which interactions are most important in determining the observed locations of the lipids around AQP0? And, does the presence of the protein influence lipids beyond the first shell of annular lipids? These questions were addressed by the simulation studies that we summarize in this Section. In addition, we review a simulation study that examined the effects of the presence of cholesterol on the interactions and dynamics of lipids in the vicinity of AQP0. In the first computational study that examined lipid interactions with AQP0, O’Connor and Klauda carried out MD simulations of AQP0 in a pure DMPC bilayer (for 25 ns) and a bilayer containing 48% DMPC and 52% cholesterol (for 100 ns).148 The authors claimed that most of the protein−lipid interactions observed in their simulations were consistent with the crystal structures, but they only listed a few examples. They found that the presence of cholesterol increases the average number of protein−lipid contacts via hydrogen bonding to the hydroxyl group of cholesterol or the glycerol group of DMPC (4.9 in the mixed bilayer vs 3.8 in the pure DMPC bilayer), and increases the hydrogen-bonding lifetimes by a factor of 2 to 5. In addition to hydrogen bonding, ring stacking interactions of cholesterol with aromatic side chains on AQP0 were observed, and face−face stacking was found to be more prevalent than edge-face stacking. The observed differences in protein−lipid interactions between pure DMPC and the two-component lipid mixture underscore the need to move to more complex lipid mixtures to characterize the interactions of membrane proteins with lipids in their native membranes.

restriction of the overall conformational dynamics of the AQP0 tetramer and strong correlations between neighboring subunits, both of which would be consistent with the cooperative response at high Ca2+ concentration observed in experiments (see Subsection 3.4). Experimental studies reviewed in Subsection 3.2 indicate that the water permeability of AQP0 is also regulated by the extracellular pH (e.g., as shown in Figure 3B, the Pf of bovine AQP0 increases when the pH is lowered from 7 to 6.5) and implicate H40 as a key player in conferring pH sensitivity. At neutral pH, a solvent-exposed histidine side chain (such as that of H40) is expected to be uncharged, but as the pH is lowered, it becomes protonated and hence carries a positive charge. Mechanistic details of how the protonation of H40, which is on an extracellular loop (loop A; Figure 2), influences water permeability are lacking, in part because the regulation of AQP0 permeability by pH has received very little attention from the simulation community. Its investigation would require protonating H40 (and possibly also other solvent exposed histidine side chains) in MD simulations. As far as we are aware, this has yet to be done. However, in their ∼100 ns timescale MD simulations of the junctional and nonjunctional forms of AQP0 with uncharged H40, Jensen et al. made an observation that hinted at a role for H40 in the pH regulation of AQP0 water permeability.131 Specifically, Jensen et al. observed that loop A (Figure 2) underwent a slow (∼100 ns timescale) transition between two conformations; in one of the conformations (“closed”), loop A obstructed water entry into the lumen. The authors speculated that if H40 was protonated, electrostatic repulsion with the side chain of a nearby arginine residue (R33) would shift the conformational equilibrium of loop A away from the closed conformation. This hypothesis remains to be tested by performing a simulation of AQP0 with H40 protonated. 5.4. Simulation Studies of AQP0−Lipid Interactions

The membranes of lens fiber cells are composed primarily of cholesterol and phospholipids, including sphingomyelin, phosphatidylcholines, and phosphatidylethanolamines.142,143 The concentration of cholesterol, the most abundant lipid in fiber cell membranes, varies within the lens; the ratio of S


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In a follow-up of the study summarized in the previous paragraph, Briones et al. examined several additional aspects of AQP0−DMPC interactions, including the effects of temperature and lipid phase.149 They found that lowering the temperature or inducing the lipid gel phase (by changing water models) did not significantly perturb the localization of the annular lipids. Surprisingly, they found that lipid immobilization did not significantly affect protein mobility but, consistent with their previous observations,147 restricting protein mobility led to increased localization of the annular lipids. Analysis of area-per-lipid and acyl chain order parameters as a function of the distance of a lipid from the protein surface revealed the coexistence of regions of gel and fluid phase lipids in the vicinity of AQP0, and corresponding measurements of the interphosphate distance and hydrophobic thickness showed that the lipids are more extended (i.e., the bilayer is thicker) next to the protein surface than lipids far from the protein surface. The authors posited that the ordering and thickening of the annular lipids is a mechanism by which the DMPC lipids accommodate their hydrophobic mismatch with AQP0. This has potential functional relevance, as it was subsequently shown using a combination of experiments and MD simulations that the water permeability of AQP4 decreased with decreasing bilayer hydrophobic thickness.150 In a computational tour de force, Stansfield et al. used a combination of coarse-grained (CG) and atomistic MD simulations to examine the interactions of lipids with 40 different members of the aquaporin and aquaglyceroporin family, including AQP0.151 The CG model they employed maps roughly four heavy atoms to one CG “bead”, thus greatly reducing the number of particles in the simulation, and enabling relatively long simulation times on relatively large systems. Stansfield et al. used CG simulations to equilibrate their systems, which included single AQPs in self-assembled bilayers and, for selected AQPs (including AQP0) in twodimensional crystals, for 1 μs. After the equilibration, the CG representations were converted to atomistic representations, and 100 ns atomistic MD trajectories were generated. Consistent with Aponte-Santamaria,147 Stansfield et al. found that the protein−phospholipid interactions sampled in their MD simulations of isolated AQPs in bilayers were similar to those observed in electron crystallographic structures of twodimensional crystals.38,146,152 Their survey of phospholipid interactions with 40 different AQPs revealed that, although there is some conservation of specific protein−lipid interactions across the AQPs, there is no evidence for highspecificity binding sites on the surfaces of AQPs. These observations led the authors to posit that, rather than being surrounded by a fixed annular shell of lipids, bulk lipids exchange with annular lipids at conserved locations on the protein surface.

All three of the questions posed in the introductory paragraph of this Section were addressed by AponteSantamaria et al.,147 who carried out MD simulations of a single AQP0 tetramer in an extended DMPC bilayer and four AQP0 tetramers, each surrounded by a single shell of DMPC lipids, in a square array that is representative of the twodimensional crystals used in the electron crystallography experiments.38,146 The MD trajectories were used to calculate lipid density maps that were compared with the locations of the DMPC lipids in the crystallographic structures. The significant extent of overlap of the MD densities from the single AQP0 tetramer trajectory with the crystallographic lipids suggested that the locations of the crystallographic lipids are physiologically relevant. Not surprisingly, lipids located between monomers in the AQP0 array were found to have a stronger degree of localization and alignment than those surrounding the single AQP0 tetramer. By introducing in silico mutations (to alanine) at residues that protein−lipid interaction energy calculations indicated strong interactions, the authors showed that electrostatic interactions played only a minor role in attracting lipids to the AQP0 surface. Rather, they found that the acyl chains define the lipid positions, and that protein mobility plays a significant role, with lipids being more strongly localized around less mobile regions of the protein surface. Far from the protein surfaces, the lipid densities at a given depth in the bilayer were uniform, indicating bulk-like behavior. Between the annular and bulklike regions, density inhomogeneities persisted for up to a few lipid shells around particular regions of the protein surface with noteworthy differences in the two leaflets of the bilayer (Figure 19).

6. CONCLUSIONS AND FUTURE DIRECTIONS The study of aquaporins in general and AQP0 in particular is paradigmatic of a modern approach in membrane biophysics in which insights drawn from high-resolution membrane protein structures and MD simulations initiated from those structures in a membrane environment are used in concert with results from in vitro experiments to provide mechanistic hypotheses for protein function. Owing to this approach, we now have a clear understanding of the molecular underpinnings of aquaporin water permeation and selectivity against protons and other ions, as well as the unique structural features that

Figure 19. Lipid density around an AQP0 monomer beyond the annular lipid shell, recovered from the simulation of a single tetramer embedded in a DMPC lipid bilayer without the crystallographic lipids. The color maps represent lateral projections (onto the xy membrane plane) of the lipid density at the different z positions indicated on the AQP0 monomer (white). Projections were taken at the average z positions of the center of masses (COM) of the indicated lipid groups (for both leaflets, upper and lower maps) and the AQP0 monomer (middle map). Reproduced with permission from ref 147. Copyright 2012 National Academy of Sciences, United States. T


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confer mammalian AQP0 with a comparatively low water permeability. It is now well-established that AQP0 is absolutely required for proper eye lens development and lens transparency. To understand the specific roles of AQP0 function in lens homeostasis, experimentalists are taking advantage of the diverse properties of AQP0 fish orthologues. What makes some of these orthologues of AQP0 conduct water at faster rates than mammalian AQP0 remains to be elucidated, presumably with the help of MD simulations. Water permeability through AQP0 is regulated by Ca2+ and pH. A combination of structural analyses and MD simulations was instrumental in providing a mechanistic hypothesis for Ca2+ regulation via complexation of AQP0 with CaM that has been validated by in vitro experiments. The structural model of the AQP0−CaM is consistent with the experimental evidence indicating that Ca2+ regulation is cooperative, but a specific structural and/or dynamical mechanism for this cooperativity has yet to be formulated. Residues involved in the pH sensitivity of AQP0 water permeability have been identified. The next challenge for MD simulations is to advance mechanistic hypotheses for regulation of AQP0 by pH that can be tested using mutagenesis and in vitro permeability assays. The dependence of AQP0 water permeability on the membrane lipid composition observed in vitro experiments remains a puzzle that could be solved in future MD simulation studies that take into account the specific lipid compositions used in the experiments that serve to validate simulation results. Moreover, MD simulations are particularly well-suited to generate hypotheses regarding a specific role of the eye lens membrane lipid composition on the regulation of AQP0 water permeability

Institute for Genomics and Bioinformatics working with Doug Tobias and Steve White, where he was supported by a bioinformatics training program from the National Institutes of Health from 2005 to 2007. He is currently a project scientist in the Department of Chemistry at the University of California, Irvine. Alfredo studies structure, dynamics, and function of proteins, membranes, and soft interfaces using theoretical and computational methods. Doug Tobias received B.S. and M.S. degrees in Chemistry from the University of California, Riverside, in 1984 and 1985, respectively. He received his Ph.D. in Chemistry and Biophysics under the direction of Professor Charles L. Brooks III at Carnegie Mellon University in 1991, where he was a National Institutes of Health predoctoral trainee from 1987−1990. Doug was a postdoctoral researcher in the lab of Professor Michael Klein in the chemistry department at the University of Pennsylvania from 1991−1995, where he was supported by a National Research Service Award from the National Institutes of Health from 1991−1994. He was a guest researcher at the National Institute of Standards and Technology from 1995−1997, and he joined the faculty of the Department of Chemistry at the University of California, Irvine, in 1997. Doug uses molecular simulation techniques to study protein dynamics, solvation, and protein−protein and protein−lipid interactions in aqueous solution and membranes. Doug is a fellow of the American Association for the Advancement of Science, the American Chemical Society, and the American Physical Society. He is also the recipient of the Theoretical Chemistry Award from the Physical Chemistry Division of the American Chemical Society and the Soft Matter and Biophysical Chemistry Award from the Royal Society of Chemistry.

ACKNOWLEDGMENTS This work was supported by grant R01 EY005661 from the National Eye Institute of the National Institutes of Health. We are grateful to the collaborators and coworkers that have contributed to the authors’ own research in the field, including Grischa Chandy, Daniel Clemens, James Fields, Tamir Gonen, Matthias Heyden, Kaitlan Kalman, Allan Miller, Karin Németh-Cahalan, Steve Reichow, Tom Schilling, Irene Vorontsova, and Guido Zampighi.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]; Tel.: 949-824-5835. *E-mail: [email protected]; Tel.: 949-824-4295. ORCID

ABBREVIATIONS ADH antidiuretic hormone AKAP2 A-kinase anchoring protein 2 AQP aquaporin AQP0 aquaporin 0 AQP1 aquaporin 1 AQP4 aquaporin 4 AQPZ aquaporin Z ar/R aromatic/arginine BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid bAPQ0 bovine aquaporin 0 bR bacteriorhodopsin CaM calmodulin cDNA cDNA CHIP channel-like intrinsic protein DEPC diethylpyrocarbonate DMPC dipalmitoylphosphatidylcholine DOPC dioleoylphosphatidylcholine EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′tetraacetic acid EM electron microscopy FMA functional mode analysis

J. Alfredo Freites: 0000-0001-5842-7443 Douglas J. Tobias: 0000-0002-6971-9828 Notes

The authors declare no competing financial interest. Biographies Jim Hall received a B.A. in Astronomy from Pomona College in 1963. He received his M.S. and Ph.D. in Physics from the University of California, Riverside in 1965 and 1968, respectively. Jim was a postdoctoral fellow at Caltech with Carver Mead, Max Delbrück, and Sunney Chan from 1970 to 1974, where he was one of the first people to observe single ion channel activity in planar lipid bilayers. He was an Assistant Professor at Duke University in the Department of Physiology and Pharmacology from 1974 to 1978 when he moved to the University of California, Irvine as Associate Professor. Jim’s research interests have moved from ion channels through gap junctions to his current focus, the properties of aquaporin zero and their biological consequences in development and homeostasis of the lens of the eye. Jim is a fellow of the American Association for the Advancement of Science. Alfredo Freites earned his Ph.D. in physics at the University of California, Irvine, in 2004. He was a postdoctoral fellow at the UCI U


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glycerol uptake facilitator protein lysophosphatidic acid molecular dynamics major intrinsic protein aquaporin 0 from Fundulus heteroclitus nuclear magnetic resonance glutamine-proline-alanine phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol protein kinase A palmitoyl oleoyl phosphatidylserine sphingomyelin wild-type

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Experimental and Simulation Studies of Aquaporin 0 Water

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