Aquaporin-0 Research Articles

Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling

Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10min after incubation in medium containing 1000μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0.AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner.The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.

Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

Aquaporin-0 Interacts with the FERM Domain of Ezrin/Radixin/Moesin Proteins in the Ocular Lens

Aquaporin 0 (AQP0) is the major intrinsic protein in the lens and is essential for establishing proper fiber cell structure and organization. Cytoskeletal proteins that directly interact with the C terminus of AQP0 are identified herein. The water-insoluble fraction of lens fiber cells was chemically cross-linked, and cross-linked peptides with the C terminus of AQP0 were identified by mass spectrometry. Coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments were used to confirm the protein-protein interaction. Unexpectedly, AQP0 was found to directly associate with ezrin/radixin/moesin (ERM) family members, proteins that are involved in linkage of actin filaments to the plasma membrane. Cross-linked peptides were detected between AQP0 and degenerate sequences of ezrin and radixin; however, AQP0 interaction with ezrin is believed to play a more significant function in the lens because of its higher level of expression and observed ezrin-specific cross-linking. The interaction was found to occur between the C terminus of AQP0 and subdomains F1 and F3 of ERM proteins. The interaction between AQP0 and ezrin was confirmed by coimmunoprecipitation and AQP0 C-terminal peptide pulldown experiments. Considering the important known functions of the cellular actin cytoskeleton in fiber cell differentiation, the interaction of AQP0 and ERM proteins may play an important role in fiber cell morphology, elongation, and organization.

The Common Modification in αA-Crystallin in the Lens, N101D, Is Associated with Increased Opacity in a Mouse Model

To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human αA-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as αAN101D-transgene and is considered to be "deamidated" in this study. Mice expressing αAN101D-transgene are referred to here CRYAA(N101D) mice. All of the lines showed the expression of αAN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) αA-transgene (referred to as CRYAA(WT) mice), the lenses of CRYAA(N101D) mice showed (a) altered αA-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT αA-crystallin, the lenses containing the deamidated αA-crystallin also showed an aggregation of αA-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated αA-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.

The role of ascorbic acid transporter in the lens of streptozotocin-induced diabetic rat

Ascorbic acid (AA) is found in high concentrations in the lens and protects the lens from photochemical events such as photo-oxidation. Diabetes reduces the concentration of AA in the lens, and it has been suggested that AA supplementation may rescue lens AA in diabetic patients. In the present study, the effects of dietary supplementation with AA were studied to assess changes in AA concentration and AA transporter expression in the lenses of streptozotocin (STZ)-induced diabetic rats. AA was lower in the aqueous humors and lenses of diabetic rats than in control rats, and low AA levels could be normalized with dietary AA supplementation. The sodium-dependent ascorbic acid transporter2, SVCT2, is transporter of AA, and glucose transporter 1 and 3 (GLUT1 and GLUT3) are transporters of dehydroascobic acid. Transcription of GLUT1 and GLUT3 was not changed as a result of diabetes or AA treatment, and the SVCT2 was increased in diabetic rats and normalized by AA supplementation in diabetic rats. Aquaporin 0 (AQP0) expression was increased in diabetic rats and by AA treatment. The rate of AA transport of vesicle originally from diabetic lens and that with AA treatment were greater than that from control rats with or without AA treatment. These results suggest that ascorbates are transported by AQP0 as well as by SVCT2, GLUT1 and GLUT3, and that supplementation with AA may help prevent diabetic cataracts.

Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50

Both connexin 50 (Cx50) and aquaporin 0 (AQP0) have important roles in lens development and homeostasis, and their mutations are associated with human congenital cataracts. We have previously shown that Cx50 directly interacts with AQP0. Here, we demonstrate the importance of the Cx50 intracellular loop (IL) domain in mediating the interaction with AQP0 in the lens in vivo. AQP0 significantly increased (~20-30%) the intercellular coupling and conductance of Cx50 gap junctions. However, this increase was not observed when the IL domain was replaced with those from other lens connexins. The Cx50-AQP0 interaction had no effect on Cx50 hemichannel function. A fusion protein containing three extracellular loop domains of AQP0 efficiently blocked the cell-to-cell adhesion of AQP0 and attenuated the stimulatory effect of AQP0 on Cx50 gap junction conductance. These data suggest that the specific interaction between Cx50 and AQP0 enhances the coupling of Cx50 gap junctions, but not hemichannels, through the cell adhesion function of AQP0. This result establishes a physiological role of AQP0 in the functional regulation of gap junction channels.

Mutations at key pore-lining positions differentiate the water permeability of fish lens aquaporin from other vertebrates

Aquaporin-0 (AQP0) is the major integral membrane protein of lens fiber cell and helps to maintain lens transparency by mediating inter-cell adhesion. To shed light on the unexpected higher water transport efficiency of killifish AQP0 as compared to mammalian orthologues, we performed a comparative analysis of all available AQP0 sequences and built 3D-models for representatives of different vertebrate classes. The analysis shows that air-living organisms evolved specific mutations at pore-lining positions to modulate the AQP0 water transport efficiency while maintaining the correct tertiary/quaternary arrangement to allow the formation of “thin junctions” between lens fiber cells. We conclude that the low permeability of mammalian AQP0 is required not to promote cell adhesion, but to modulate the water balance in a dry environment.

The role of ascorbic acid transporter in the lens of streptozotocin-induced diabetic rat

Ascorbic acid (AA) is found in high concentrations in the lens and protects the lens from photochemical events such as photo-oxidation. Diabetes reduces the concentration of AA in the lens, and it has been suggested that AA supplementation may rescue lens AA in diabetic patients. In the present study, the effects of dietary supplementation with AA were studied to assess changes in AA concentration and AA transporter expression in the lenses of streptozotocin (STZ)-induced diabetic rats. AA was lower in the aqueous humors and lenses of diabetic rats than in control rats, and low AA levels could be normalized with dietary AA supplementation. The sodium-dependent ascorbic acid transporter2, SVCT2, is transporter of AA, and glucose transporter 1 and 3 (GLUT1 and GLUT3) are transporters of dehydroascobic acid. Transcription of GLUT1 and GLUT3 was not changed as a result of diabetes or AA treatment, and the SVCT2 was increased in diabetic rats and normalized by AA supplementation in diabetic rats. Aquaporin 0 (AQP0) expression was increased in diabetic rats and by AA treatment. The rate of AA transport of vesicle originally from diabetic lens and that with AA treatment were greater than that from control rats with or without AA treatment. These results suggest that ascorbates are transported by AQP0 as well as by SVCT2, GLUT1 and GLUT3, and that supplementation with AA may help prevent diabetic cataracts.

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: Effects on lens transparency

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0 −/−) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1 +/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1 +/+/AQP0 −/−) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.

Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals

We have previously described the interactions of aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 Å structure of AQP0 in two-dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Comparison of the two structures shows that AQP0 does not adapt to the different length of the acyl chains in EPLs and that the distance between the phosphodiester groups in the two leaflets of the DMPC and EPL bilayers is almost identical. The EPL headgroups interact differently with AQP0 than do those of DMPC, but the acyl chains in the EPL and DMPC bilayers occupy similar positions. The interactions of annular lipids with membrane proteins seem to be driven by the propensity of the acyl chains to fill gaps in the protein surface. Interactions of the lipid headgroups may be responsible for the specific interactions found in tightly bound lipids but seem to have a negligible effect on interactions of generic annular lipids with membrane proteins.

Electrostatics of aquaporin and aquaglyceroporin channels correlates with their transport selectivity

Aquaporins are homotetrameric channel proteins, which allow the diffusion of water and small solutes across biological membranes. According to their transport function, aquaporins can be divided into "orthodox aquaporins", which allow the flux of water molecules only, and "aquaglyceroporins", which facilitate the diffusion of glycerol and other small solutes in addition to water. The contribution of individual residues in the pore to the selectivity of orthodox aquaporins and aquaglyceroporins is not yet fully understood. To gain insights into aquaporin selectivity, we focused on the sequence variation and electrostatics of their channels. The continuum Poisson-Boltzmann electrostatic potential along the channel was calculated and compared for ten three-dimensional-structures which are representatives of different aquaporin subfamilies, and a panel of functionally characterized mutants, for which high-accuracy three-dimensional-models could be derived. Interestingly, specific electrostatic profiles associated with the main selectivity to water or glycerol could be identified. In particular: (i) orthodox aquaporins showed a distinctive electrostatic potential maximum at the periplasmic side of the channel around the aromatic/Arg (ar/R) constriction site; (ii) aquaporin-0 (AQP0), a mammalian aquaporin with considerably low water permeability, had an additional deep minimum at the cytoplasmic side; (iii) aquaglyceroporins showed a rather flat potential all along the channel; and (iv) the bifunctional protozoan PfAQP had an unusual all negative profile. Evaluation of electrostatics of the mutants, along with a thorough sequence analysis of the aquaporin pore-lining residues, illuminated the contribution of specific residues to the electrostatics of the channels and possibly to their selectivity.

Dynamic and energetic mechanisms for the distinct permeation rate in AQP1 and AQP0

Despite sharing overall sequence and structural similarities, water channel aquaporin 0 (AQP0) transports water more slowly than other aquaporins. Using molecular dynamics simulations of AQP0 and AQP1, we find that there is a sudden decrease in the distribution profile of water density along the pore of AQP0 in the region of residue Tyr23, which significantly disrupts the single file water chain by forming hydrogen bond with permeating water molecules. Comparisons of free-energy and interaction-energy profiles for water conduction between AQP0 and AQP1 indicate that this interruption of the water chain causes a huge energy barrier opposing water translocation through AQP0. We further show that a mutation of Tyr23 to phenylalanine leads to a 2- to 4-fold enhancement in water permeability of AQP0, from (0.5 ± 0.2) × 10 − 14 cm 3s − 1 to (1.9 ± 0.6) × 10 − 14 cm 3s − 1 . Therefore, Tyr23 is a dominate factor leading to the low water permeability in AQP0.

Sorting of Lens Aquaporins and Connexins into Raft and Nonraft Bilayers: Role of Protein Homo-Oligomerization

Two classes of channel-forming proteins in the eye lens, the water channel aquaporin-0 (AQP-0) and the connexins Cx46 and Cx50, are preferentially located in different regions of lens plasma membranes ( 1,2). Because these membranes contain high concentrations of cholesterol and sphingomyelin, as well as phospholipids such as phosphatidylcholine with unsaturated hydrocarbon chains, microdomains (rafts) form in these membranes. Here we test the hypothesis that sorting into lipid microdomains can play a role in the disposition of AQP-0 and the connexins in the plane of the membrane. For both crude membrane fractions and proteoliposomes composed of lens proteins in phosphatidylcholine/sphingomyelin/cholesterol lipid bilayers, detergent extraction experiments showed that the connexins were located primarily in detergent soluble membrane (DSM) fractions, whereas AQP-0 was found in both detergent resistant membrane and DSM fractions. Analysis of purified AQP-0 reconstituted in raft-containing bilayers showed that the microdomain location of AQP-0 depended on protein/lipid ratio. AQP-0 was located almost exclusively in DSMs at a 1:1200 AQP-0/lipid ratio, whereas ∼50% of the protein was sequestered into detergent resistant membranes at a 1:100 ratio, where freeze-fracture experiments show that AQP-0 oligomerizes ( 3). Consistent with these detergent extraction results, confocal microscopy images showed that AQP-0 was sequestered into raft microdomains in the 1:100 protein/lipid membranes. Taken together these results indicate that AQP-0 and connexins can be segregated in the membrane by protein-lipid interactions as modified by AQP-0 homo-oligomerization.

MALDI Imaging Mass Spectrometry of Integral Membrane Proteins from Ocular Lens and Retinal Tissue

A tissue preparation protocol for MALDI (matrix-assisted laser desorption/ionization) imaging mass spectrometry of integral membrane proteins was developed using ocular lens and retinal tissues as model samples. Frozen bovine and human lenses were cryosectioned equatorially or axially at -20 degrees C into 20 mum-thick tissue sections. Lens sections were mounted onto gold-coated MALDI targets by methanol soft-landing to maintain tissue integrity. Tissue sections underwent extensive water washing to deplete the samples of highly abundant water-soluble proteins. Automated matrix deposition was achieved using an acoustic reagent multispotter, with sinapinic acid as matrix and high percentage acetonitrile as solvent, with a center-to-center spot spacing of 200-300 mum. Molecular images of full-length Aquaporin-0 (AQP0) and its most abundant truncation products were obtained from mass spectral data acquired across whole bovine and human lens sections. In equatorial and axial sections of bovine lenses, full-length AQP0 was detected throughout the lens. A truncation product corresponding to AQP0 (1-260) was detected in the bovine lens core at low abundance. In axial lens sections, no antero-posterior variation was detected. In 11 year-old human lens sections, full-length AQP0 was most abundant in the lens periphery, but was detected throughout the lens. The major truncation product, consisting of AQP0 residues 1-246, was absent from the lens periphery and increased in abundance in the lens core. This tissue preparation protocol was then applied to image the distribution of the G-protein coupled receptor, opsin, in the rabbit retina. This protocol has expanded the variety of target analytes which can be detected by MALDI imaging mass spectrometry to include intact integral membrane proteins.

Electron Microscopy of AQP0-mediated Membrane Junctions

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fiber cells in the lens. We describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic N- and C-termini. The pore in junctional AQP0 appears to be closed and retains only three water molecules in the center of the pore, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. This made it possible to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers and to describe non-specific lipid-protein interactions.

Protein aging: Truncation of aquaporin 0 in human lens regions is a continuous age-dependent process

The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2 kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40–50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide ‘spacer’ may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age.

Malformation of junctional microdomains in cataract lens membranes from a type II diabetes patient

In eye core lens membranes, aquaporin-0 (AQP0) and connexins (Cx) form together well-structured supramolecular assemblies, the junctional microdomains, in which they assure water, ion, metabolite, and waste transport. Additionally, they mediate cell-cell adhesion-forming thin junctions (AQP0) and gap junctions (Cx). We have used atomic force microscopy and biochemical methods to analyze and compare the structure of junctional microdomains in human cataract lens membranes from a type II diabetes patient and healthy lens membranes from calf. A healthy intercellular junctional microdomain consists in average of approximately 150 tetragonally arranged (a = b = 65.5 A, gamma = 90 degrees) AQP0 tetramers surrounded by densely packed non-ordered connexon channels. Gap-junction connexons act as lineactants inside the membrane and confine AQP0 in the junctional microdomains. In the diabetic cataract lens, connexons were degraded, and AQP0 arrays are malformed. We conceptualize that absence of connexons lead to breakdown of cell nutrition.

Differentiation-dependent modification and subcellular distribution of aquaporin-0 suggests multiple functional roles in the rat lens

Using immunohistochemistry and mass spectrometry, differentiation-dependent changes in the subcellular distribution and processing of aquaporin-0 (AQP0) have been mapped in the rat lens. Sections labelled with C-terminal tail AQP0 antibodies yielded two concentric rings of labelling with minimal signal in the lens core. The rings were separated by a transient zone of decreased labelling located prior to the transition of differentiating fiber (DF) cells into mature denucleated fiber (MF) cells. Mass spectrometry showed that the loss of core labelling was due to AQP0 cleavage, while the transient loss of labelling was more likely caused by masking of the antibody epitope. AQP0 subcellular distribution changed with radial distance into the lens. In peripheral DF cells, AQP0 was found throughout both broad and narrow side membranes. In deeper-lying DF cells, AQP0 aggregated into plaque-like structures located on the broad sides. This shift occurred prior to the transient loss of AQP0 signal, and coincided with formation of broad-side membrane invaginations between adjacent fiber cells to which filensin, a known binding partner of AQP0, was also localized. After nuclei loss, AQP0 was once again distributed throughout MF cell membranes. In the absence of protein synthesis, the observed subcellular redistribution of AQP0 in DF and subsequent cleavage of AQP0 in MF are suggestive of a switch in the function of AQP0 from a water channel to a junctional protein.

Dynamic control of slow water transport by aquaporin 0: Implications for hydration and junction stability in the eye lens

Aquaporin 0 (AQP0), the most abundant membrane protein in mammalian lens fiber cells, not only serves as the primary water channel in this tissue but also appears to mediate the formation of thin junctions between fiber cells. AQP0 is remarkably less water permeable than other aquaporins, but the structural basis and biological significance of this low permeability remain uncertain, as does the permeability of the protein in a reported junctional form. To address these issues, we performed molecular dynamics (MD) simulations of water transport through membrane-embedded AQP0 in both its (octameric) junctional and (tetrameric) nonjunctional forms. From our simulations, we measured an osmotic permeability for the nonjunctional form that agrees with experiment and found that the distinct dynamics of the conserved, lumen-protruding side chains of Tyr-23 and Tyr-149 modulate water passage, accounting for the slow permeation. The junctional and nonjunctional forms conducted water equivalently, in contrast to a previous suggestion based on static crystal structures that water conduction is lost on junction formation. Our analysis suggests that the low water permeability of AQP0 may help maintain the mechanical stability of the junction. We hypothesize that the structural features leading to low permeability may have evolved in part to allow AQP0 to form junctions that both conduct water and contribute to the organizational structure of the fiber cell tissue and microcirculation within it, as required to maintain transparency of the lens.