Structural determinants of proton blockage in aquaporins

Aquaporins are an important class of membrane channels selective for water and linear polyols but impermeable to ions, including protons. Recent computational studies have revealed that the relay of protons through the water-conduction pathway of aquaporin channels is opposed by a substantial free energy barrier peaking at the signature NPA motifs. Here, free-energy simulations and continuum electrostatic calculations are combined to examine the nature and the magnitude of the contribution of specific structural elements to proton blockage in the bacterial glycerol uptake facilitator, GlpF. Potential of mean-force profiles for both hop and turn steps of structural diffusion in the narrow pore are obtained for artificial variants of the GlpF channel in which coulombic interactions between the pore contents and conserved residues Asn68 and Asn203 at the NPA signature motifs, Arg206 at the selectivity filter, and the peptidic backbone of the two half-helices M3 and M7, which are arranged in head-to-head fashion around the NPA motifs, are turned off selectively. A comparison of these results with electrostatic energy profiles for the translocation of a probe cation throughout the water permeation pathway indicates that the free-energy profile for proton movement inside the narrow pore is dominated by static effects arising from the distribution of charged and polar groups of the channel, whereas dielectric effects contribute primarily to opposing the access of H+ to the pore mouths (desolvation penalty). The single most effective way to abolish the free-energy gradients opposing the movement of H+ around the NPA motif is to turn off the dipole moments of helices M3 and M7. Mutation of either of the two NPA Asn residues to Asp compensates for charge-dipole and dipole-dipole effects opposing the hop and turn steps of structural diffusion, respectively, and dramatically reduces the free energy barrier of proton translocation, suggesting that these single mutants could leak protons.     

Hydroxide and proton migration in aquaporins

Hypothetical hydroxide and proton migration along the linear water chain in Aquaporin GlpF from Escherichia coli are studied by ab initio Car-Parrinello molecular dynamics simulations. It is found that the protein stabilizes a bipolar single file of water. The single file features a contiguous set of water-water hydrogen bonds in which polarization of the water molecules vary with position along the channel axis. Deprotonation of the water chain promotes the reorientation of water molecules while the hydroxide ion rapidly migrates by sequentially accepting protons from the neighboring water molecules. The hydroxide ion is not attracted by a conserved, channel-lining arginine residue, but is immobilized at two centrally located, conserved Asparagine-Proline-Alanine motifs where fourfold coordination stabilizes the ion. Hydroxide transition from the channel vestibules into the channel lumen is strongly influenced by electrostatic coupling to two conserved oppositely aligned macrodipoles. This suggests that the macrodipole's negative poles play a role in preventing hydroxide ions from entering into the channel's inner vestibules. Water protonation within the lumen facilitates water reorientation and subsequent proton expelling occurs. In the periplasmic half-channel, expelling occurs via the Grotthuss mechanism. Protonation within the cytoplasmic half-channel implies wire-breakage at the Asn-Pro-Ala motifs. The proton is here diffusively rejected as (H(5)O(2))(+).     

Water and ion permeation in bAQP1 and GlpF channels: a kinetic Monte Carlo study

The kinetic Monte Carlo reaction-path-following technique is applied to determine the lowest-energy water pathway and the coordinating amino acids in bAQP1 and GlpF channels, both treated as rigid. In bAQP1, water molecules pass through the pore between the asparagine-proline-alanine (NPA) and selectivity filter (SF) sites one at a time. The water chain is interrupted at the SF where one water forms three stable hydrogen bonds with protein atoms. In this SF, water's conformation depends on the protonation locus of H182. In GlpF, two water molecules bond simultaneously to the NPA asparagines and pass through the SF in zigzag fashion. No water single-file forms in rigid GlpF. To accommodate a single file of waters requires narrowing the GlpF pore. Our results reveal that in both proteins a proposed bipolar water arrangement is thermally disrupted in the NPA region, especially in the cytoplasmic part of the pore. The equilibrium hydrogen-bonded chain is occasionally interrupted in the hydrophobic zones adjacent to the NPA motifs. The permeation of alkali cations through bAQP1 and GlpF is barred due to a large free-energy barrier in the NPA region as well as a large energy barrier blocking entry from the cytoplasm. Permeation of halides is prevented due to two large energy barriers in the cytoplasmic and periplasmic pores as well as a large free-energy barrier barring entry from the periplasm. Our results, based on modeling charge permeation, support an electrostatic rather than orientational basis for proton exclusion. Binding within the aquaporin pore cannot compensate sufficiently for dehydration of the protonic charge; there is also an electrostatic barrier in the NPA region blocking proton transport. The highly ordered single file of waters, which is drastically interrupted at the SF of bAQP1, may also contribute to proton block.     

The mechanism of glycerol conduction in aquaglyceroporins.

BACKGROUND: The E. coli glycerol facilitator, GlpF, selectively conducts glycerol and water, excluding ions and charged solutes. The detailed mechanism of the glycerol conduction and its relationship to the characteristic secondary structure of aquaporins and to the NPA motifs in the center of the channel are unknown. RESULTS: Molecular dynamics simulations of GlpF reveal spontaneous glycerol and water conduction driven, on a nanosecond timescale, by thermal fluctuations. The bidirectional conduction, guided and facilitated by the secondary structure, is characterized by breakage and formation of hydrogen bonds for which water and glycerol compete. The conduction involves only very minor changes in the protein structure, and cooperativity between the GlpF monomers is not evident. The two conserved NPA motifs are strictly linked together by several stable hydrogen bonds and their asparagine side chains form hydrogen bonds with the substrates passing the channel in single file. CONCLUSIONS: A complete conduction of glycerol through the GlpF was deduced from molecular dynamics simulations, and key residues facilitating the conduction were identified. The nonhelical parts of the two half-membrane-spanning segments expose carbonyl groups towards the channel interior, establishing a curve-linear pathway. The conformational stability of the NPA motifs is important in the conduction and critical for selectivity. Water and glycerol compete in a random manner for hydrogen bonding sites in the protein, and their translocations in single file are correlated. The suggested conduction mechanism should apply to the whole family.     

The mechanism of proton exclusion in the aquaporin-1 water channel

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.     

Molecular dynamics study of aquaporin-1 water channel in a lipid bilayer

The aquaporin-1 water channel was modeled in a palmitoyl-oleoyl-phosphatidyl-choline lipid bilayer, by means of molecular dynamics simulations. Interaction of the protein with the membrane and inter-monomer interactions were analyzed. Structural features of the channel important for its biological function, including the Asn-Pro-Ala (NPA) motifs, and the diffusion of water molecules into the channels, were investigated. Simulations revealed the formation of single file water inside the channels for certain relative positions of the NPA motifs.     

Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1

Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone collective macrodipoles that create an environment favoring a directionally discontinuous channel hydrogen-bonded water chain and a large electrostatic barrier. The SF domain contributes to the proton permeation barrier by a spatial restriction mechanism and direct electrostatic interactions. To further explore these various effects, the free-energy barriers and the maximum cation conductance for the permeation of various cations through the AQP1-R195V and AQP1-R195S mutants are predicted computationally. The cations studied included the hydrated excess proton that utilizes the Grotthuss shuttling mechanism, a model “classical” charge localized hydronium cation that exhibits no Grotthuss shuttling, and a sodium cation. The hydrated excess proton was simulated using a specialized multi-state molecular dynamics method including a proper physical treatment of the proton shuttling and charge defect delocalization. Both AQP1 mutants exhibit a surprising cooperative effect leading to a reduction in the free-energy barrier for proton permeation around the NPA region due to altered water configurations in the SF region, with AQP1-R195S having a higher conductance than AQP1-R195V. The theoretical predictions are experimentally confirmed in wild-type AQP1 and the mutants expressed in Xenopus oocytes. The combined results suggest that the SF domain is a specialized structure that has evolved to impede proton permeation in aquaporins.     

On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives)

A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or - which might create confusions with "minus".     

Water transport in human aquaporin-4: molecular dynamics (MD) simulations

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.     

Molecular basis of proton blockage in aquaporins

Water transport channels in membrane proteins of the aquaporin superfamily are impermeable to ions, including H+ and OH-. We examine the molecular basis for the blockage of proton translocation through the single-file water chain in the pore of a bacterial aquaporin, GlpF. We compute the reversible thermodynamic work for the two complementary steps of the Grotthuss "hop-and-turn" relay mechanism: consecutive transfers of H+ along the hydrogen-bonded chain (hop) and conformational reorganization of the chain (turn). In the absence of H+, the strong preference for the bipolar orientation of water around the two Asn-Pro-Ala (NPA) motifs lining the pore over both unidirectional polarization states of the chain precludes the reorganization of the hydrogen-bonded network. Inversely, translocation of an excess proton in either direction is opposed by a free-energy barrier centered at the NPA region. Both hop and turn steps of proton translocation are opposed by the electrostatic field of the channel.     

Water wires in atomistic models of the Hv1 proton channel

The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.     

The barrier for proton transport in aquaporins as a challenge for electrostatic models: the role of protein relaxation in mutational calculations

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Dynamics and energetics of water permeation through the aquaporin channel

Structural properties of water inside bovine aquaporin-1 are investigated by molecular simulation. The calculations, which are based on the recently determined X-ray structure at 2.2 A resolution (Sui et al., Nature 2001;414:872-878), are carried out on one monomeric subunit immersed in a water-n-octane-water bilayer. Molecular dynamics (MD) simulations suggest that His182, a fully conserved residue in the channel pore, is protonated in the delta position. Furthermore, they reveal a highly ordered water structure in the channel, induced by the electrostatic properties of the protein. Multiple-steering MD simulations are used to calculate the free-energy of water diffusion. To the best of our knowledge, this represents the first free-energy calculation based on the new, high-resolution structure of the pore. The calculated barrier is 2.5 kcal/mol, and it is associated to water permeation through the Asn-Pro-Ala (NPA) region of the pore, where water molecules are only hydrogen-bonded with themselves. These findings are fully consistent with those based on the previous MD studies on the human protein (de Groot and Grubmüller, Science 2001;294:2353-2357).     

Multiple pore lining residues modulate water permeability of GlpF

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin “open state” is still considered to be a continuously open pore with water molecules permeating in a single‐file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long‐term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.     

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.     

On the origin of the electrostatic barrier for proton transport in aquaporin

The nature of the electrostatic barrier for proton transport in aquaporins is analyzed by semimacroscopic and microscopic models. It is found that the barrier is associated with the loss of the generalized solvation energy upon moving from the bulk solvent to the center of the channel. It is clarified that our solvation concept includes the effect of the protein polar groups and ionized residues. The nature of the contributions to the solvation barrier is examined by using the linear response approximation. It is found that the residues in the NPA region contribute much less than what would be deduced from calculations that do not consider the protein reorganization. It is clarified that the contributions of different structural or electrostatic elements to the solvation barrier can be established by removing these elements and examining the corresponding effect on the barrier height. Using this definition and “mutating” the NPA residues to their non-polar analogues establishes that these residues do not provide the major contribution to the solvation barrier.     

Identification of a residue in helix 2 of rice plasma membrane intrinsic proteins that influences water permeability

Molecular selection, ion exclusion, and water permeation are well known regulatory mechanisms in aquaporin. Water permeability was found to be diverse in different subgroups of plasma membrane intrinsic proteins (PIPs), even though the residues surrounding the water holes remained the same across the subgroups. Upon homology modeling and structural comparison, a conserved Ala/Ile(Val) residue difference was identified in helix 2 that affected the conformation of the NPA region and consequently influenced the water permeability. The residue difference was found to be conservative within the two subgroups of PIPs in rice as well as in other plants. Functional tests further confirmed the prediction via site-directed mutagenesis where replacement of Ala(103) or Ala(102) in respective OsPIP1;1 or OsPIP1;3 with Val yielded 7.0- and 2.2-fold increases in water transportation, and substitution of Ile(98) or Val(95) in respective OsPIP2;3 or OsPIP2;7 with Ala resulted in 73 or 52% reduction of water transportation. Based on structural analyses and molecular dynamics simulations, we proposed that the difference in water permeability was attributed to the orientation variations of helix 2 that modified water-water and water-protein interactions.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the “subcellular AQPs”. In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72 ± 0.03 × 10^− 13 cm³/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Molecular dynamics simulation and bioinformatics study on yeast aquaporin Aqy1 from Pichia pastoris

In the present study, an equilibrated system for the Aqy1 tetramer was developed, and molecular biophysics modeling showed that the Aqy1 channel was blocked by Tyr-31 in the N-terminus, which was also supported by the free energy profiles. However, bioinformatics analysis of the amino acid sequence of Aqy1 indicated this Tyr-31 is not conserved across all fungi. Analysis of the equilibrated structure showed that the central pore along the four-fold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-90, Trp-198, and Phe-202 form the narrowest part of the pore. Therefore, water molecules are not expected to translocate through the central pore, a hypothesis that we confirmed by molecular dynamics simulations. Each monomer of the Aqy1 tetramers forms a channel whose walls consist mostly of hydrophilic residues, transporting through the selectivity filter containing Arg-227, His-212, Phe-92, and Ala-221, and the two conserved Asn-Pro-Ala (NPA) motifs containing asparagines 224 and 112. In summary, not all fungal aquaporins share the same gating mechanism by a tyrosine residue in the N-terminus, and the structural analysis in the present study should aid our understanding of aquaporin structure and its functional implications.