Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0^+/−) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0^+/−/AQP1^+/−) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P_f) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA, possibly due to alteration in RING. To our knowledge, this is the first report identifying the role of AQP0 in RING development to ward off lens SA during focusing.     

Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein.

Aquaporin-0 (AQP0) is the major intrinsic protein of lens fiber cells and the founder member of the water channel gene family. Here we show that disruption of the AQP0 gene by an early transposon (ETn) element results in expression of a chimeric protein, comprised of approximately 75% AQP0 and approximately 25% ETn long terminal repeat (LTR) sequence, in the cataract Fraser (CatFr) mouse lens. Immunoblot analysis showed that mutant AQP0-LTR was similar in mass to wild-type AQP0. However, immunofluorescence microscopy revealed that AQP0-LTR was localized to intracellular membranes rather than to plasma membranes of lens fiber cells. Heterozygous CatFr lenses were similar in size to wild-type but displayed abnormal regions of translucence and light scattering. Scanning electron microscopy further revealed that mature fiber cells within the core of the heterozygous CatFr lens failed to stratify into uniform, concentric growth shells, suggesting that the AQP0 water channel facilitates the development of the unique cellular architecture of the crystalline lens.     

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.     

Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency

Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fiber cell adhesion are different in AQP0^−/−, and TgAQP1^+/+/AQP0^−/− mice that transgenically express AQP1 (TgAQP1) in fiber cells without AQP0 (AQP0^−/−). In WT, lenses were transparent with ‘Y’ sutures. Fibers contained opposite end curvature, lateral interdigitations, hexagonal shape, and were arranged as concentric growth shells. AQP0^−/− lenses were cataractous, lacked ‘Y’ sutures, ordered packing and well-defined lateral interdigitations. TgAQP1^+/+/AQP0^−/− lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fiber cells in WT whereas AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1^+/+/AQP0^−/− mice. Fiber cell AQP0 expression is required to maintain their organization, which is a requisite for lens transparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0^−/− and TgAQP1^+/+/AQP0^−/− lenses, fiber cell disorganization was evident.     

Structure of functional single AQP0 channels in phospholipid membranes

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.     

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.     

Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice

Cataract Tohoku (Cat(Tohm)) is a dominant cataract mutation that leads to severe degeneration of lens fiber cells. Linkage analysis showed that the Cat(Tohm) mutation is located on mouse chromosome 10, close to the gene for aquaporin-0 (Aqp0), which encodes a membrane protein that is expressed specifically in lens fiber cells. Sequence analysis of Aqp0 revealed a 12-bp deletion without any change in the reading frame, which resulted in a deletion of four amino acids within the second transmembrane region of the AQP0 protein. Targeted expression of the mutated Aqp0 caused lens opacity in transgenic mice, the pathological severity of which depended on the expression level of the transgene. The mutated AQP0 protein was localized to the intracellular and perinuclear spaces rather than to the plasma membranes of the lens fiber cells. The cataract phenotype of Cat(Tohm) is caused by a gain-of-function mutation in the mutated AQP0 protein and not by a loss-of-function mutation.     

Regulation of AQP0 water permeability is enhanced by cooperativity

Aquaporin 0 (AQP0), essential for lens clarity, is a tetrameric protein composed of four identical monomers, each of which has its own water pore. The water permeability of AQP0 expressed in Xenopus laevis oocytes can be approximately doubled by changes in calcium concentration or pH. Although each monomer pore functions as a water channel, under certain conditions the pores act cooperatively. In other words, the tetramer is the functional unit. In this paper, we show that changes in external pH and calcium can induce an increase in water permeability that exhibits either a positive cooperativity switch-like increase in water permeability or an increase in water permeability in which each monomer acts independently and additively. Because the concentrations of calcium and hydrogen ions increase toward the center of the lens, a concentration signal could trigger a regulatory change in AQP0 water permeability. It thus seems plausible that the cooperative modes of water permeability regulation by AQP0 tetramers mediated by decreased pH and elevated calcium are the physiologically important ones in the living lens.     

Human cataract lens membrane at subnanometer resolution

Human pathologies often originate from molecular disorders. Therefore, imaging technology as one of the bases for the identification and understanding of pathologies must provide views of single molecules at subnanometer resolution. Membrane proteins mediate many of life's most important processes, and their malfunction is often lethal or leads to severe disease. The membrane proteins aquaporin-0 (AQP0) and connexons form junctional microdomains between healthy lens core cells in which AQP0 form square arrays surrounded by connexons. Malfunction of both proteins results in the formation of cataract. We have used high-resolution atomic force microscopy (AFM) to image junctional microdomains in membranes from an individual human eye lens with senile cataract. Images at subnanometer resolution report individual helix-connecting loops of four amino acid residues on the AQP0 surface. We describe the supramolecular assembly and the conformational state of AQP0 in junctional microdomains, where a mixture of truncated junctional and full-length water channel AQP0 form square arrays. Imaging of microdomain borders revealed individual AQP0 tetramers and no associated connexon, indicating a lack of metabolite transport, waste accumulation, and enlarged regions of non-adhering membranes, causing cataract in this individual. This first high-resolution view of the membrane of this pathological human tissue provides insights into cataract pathology at the single membrane protein level, and indicates the power of the AFM as a future tool in medical imaging at subnanometer resolution.     

Co-axial association of recombinant eye lens aquaporin-0 observed in loosely packed 3D crystals

Aquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.0 A resolution. The crystal packing was determined by molecular replacement and shows that, within the cubic lattice, AQP0 tetramers are associated head-to-head along their 4-fold axes. Oligomeric states larger than the tetramer were also observed in solution by native gel electrophoresis and analytical ultracentrifugation methods. In the crystals, there are no direct contacts between octamers, and it can thus be inferred that crystalline order is mediated solely by the detergent belts surrounding the membrane protein. Across the tetramer-tetramer interface, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer structure is compared with that of the recently determined structure of truncated ovine AQP0 derived from electron diffraction of 2D crystals. Intriguingly, also in these crystals, octamers are observed, but with significantly different relative tetramer-tetramer orientations. The interactions observed in the loosely packed 3D crystals reported here may in fact represent an in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens.     

Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye

Multiple physiological fluid movements areinvolved in vision. Here we define the cellular and subcellular sitesof aquaporin (AQP) water transport proteins in human and rat eyes byimmunoblotting, high-resolution immunocytochemistry, and immunoelectronmicroscopy. AQP3 is abundant in bulbar conjunctival epithelium andglands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering thetrabecular meshwork and Schlemm's canal. Although AQP1 is plentiful inciliary nonpigmented epithelium, it is not present in ciliary pigmentedepithelium. Posterior and anterior epithelium of the iris and anteriorlens epithelium also contain significant amounts of AQP1, but AQP0(major intrinsic protein of the lens) is expressed in lens fiber cells.Retinal Müller cells and astrocytes exhibit notableconcentrations of AQP4, whereas neurons and retinal pigment epitheliumdo not display aquaporin immunolabeling. These studies demonstrateselective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocularepithelia, predicting specific roles for each in the complex networkthrough which water movements occur in the eye.

     

Developmental regulation of the direct interaction between the intracellular loop of connexin 45.6 and the C terminus of major intrinsic protein (aquaporin-0)

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.     

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.     

AQP0-LTR of the Cat Fr mouse alters water permeability and calcium regulation of wild type AQP0

Aquaporin 0 (AQP0) is the major intrinsic protein of the lens and its water permeability can be modulated by changes in pH and Ca2+. The Cataract Fraser (Cat Fr) mouse accumulates an aberrant AQP0 (AQP0-LTR) in sub-cellular compartments resulting in a congenital cataract. We investigated the interference of AQP0-LTR with normal function of AQP0 in three systems. First, we created a transgenic mouse expressing AQP0 and AQP0-LTR in the lens. Expression of AQP0 did not prevent the congenital cataract but improved the size and transparency of the lens. Second, we measured water permeability of AQP0 co-expressed with AQP0-LTR in Xenopus oocytes. A low expression level of AQP0-LTR decreased the water permeability of AQP0, and a high expression level eliminated its calcium regulation. Third, we studied trafficking of AQP0 and AQP0-LTR in transfected lens epithelial cells. At low expression level, AQP0-LTR migrated with AQP0 toward the cell membrane, but at high expression level, it accumulated in sub-cellular compartments. The deleterious effect of AQP0-LTR on lens development may be explained by lowering water permeability and abolishing calcium regulation of AQP0. This study provides the first evidence that calcium regulation of AQP0 water permeability may be crucial for maintaining normal lens homeostasis and development.     

AQP0-LTR of the Cat mouse alters water permeability and calcium regulation of wild type AQP0

Aquaporin 0 (AQP0) is the major intrinsic protein of the lens and its water permeability can be modulated by changes in pH and Ca²⁺. The Cataract Fraser (Cat^Fr) mouse accumulates an aberrant AQP0 (AQP0-LTR) in sub-cellular compartments resulting in a congenital cataract. We investigated the interference of AQP0-LTR with normal function of AQP0 in three systems. First, we created a transgenic mouse expressing AQP0 and AQP0-LTR in the lens. Expression of AQP0 did not prevent the congenital cataract but improved the size and transparency of the lens. Second, we measured water permeability of AQP0 co-expressed with AQP0-LTR in Xenopus oocytes. A low expression level of AQP0-LTR decreased the water permeability of AQP0, and a high expression level eliminated its calcium regulation. Third, we studied trafficking of AQP0 and AQP0-LTR in transfected lens epithelial cells. At low expression level, AQP0-LTR migrated with AQP0 toward the cell membrane, but at high expression level, it accumulated in sub-cellular compartments. The deleterious effect of AQP0-LTR on lens development may be explained by lowering water permeability and abolishing calcium regulation of AQP0. This study provides the first evidence that calcium regulation of AQP0 water permeability may be crucial for maintaining normal lens homeostasis and development.     

Thermal Stress Induced Aggregation of Aquaporin 0 (AQP0) and Protection by α-Crystallin via Its Chaperone Function

Aquaporin 0 (AQP0) formerly known as membrane intrinsic protein (MIP), is expressed exclusively in the lens during terminal differentiation of fiber cells. AQP0 plays an important role not only in the regulation of water content but also in cell-to-cell adhesion of the lens fiber cells. We have investigated the thermal stress-induced structural alterations of detergent (octyl glucoside)-solubilized calf lens AQP0. The results show an increase in the amount of AQP0 that aggregated as the temperature increased from 40°C to 65°C. α-Crystallin, molecular chaperone abundantly present in the eye lens, completely prevented the AQP0 aggregation at a 1∶1 (weight/weight) ratio. Since α-crystallin consists of two gene products namely αA- and αB-crystallins, we have tested the recombinant proteins on their ability to prevent thermal-stress induced AQP0 aggregation. In contrast to the general observation made with other target proteins, αA-crystallin exhibited better chaperone-like activity towards AQP0 compared to αB-crystallin. Neither post-translational modifications (glycation) nor C-terminus truncation of AQP0 have any appreciable effect on its thermal aggregation properties. α-Crystallin offers similar protection against thermal aggregation as in the case of the unmodified AQP0, suggesting that αcrystallin may bind to either intracellular loops or other residues of AQP0 that become exposed during thermal stress. Far-UV circular dichroism studies indicated a loss of αhelical structures when AQP0 was subjected to temperatures above 45°C, and the presence of α-crystallin stabilized these secondary structures. We report here, for the first time, that α-crystallin protects AQP0 from thermal aggregation. Since stress-induced structural perturbations of AQP0 may affect the integrity of the lens, presence of the molecular chaperone, α-crystallin (particularly αA-crystallin) in close proximity to the lens membrane is physiologically relevant.     

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

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 10 min after incubation in medium containing 1000 μM 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.     

Post-translational modifications of aquaporin 0 (AQP0) in the normal human lens: spatial and temporal occurrence

Because of the lack of protein turnover in fiber cells of the ocular lens, Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, undergoes extensive post-translational modification with fiber cell age. To map the distribution of modified forms of AQP0 within the lens, normal human lenses ranging in age from 34 to 38 were concentrically dissected into several cortical and nuclear sections. Membrane proteins still embedded in the membranes were digested with trypsin, and the resulting C-terminal peptides of AQP0 were analyzed by HPLC tandem mass spectrometry, permitting the identification of modifications and estimation of their abundance. Consistent with earlier reports, the major phosphorylation site was Ser 235, and the major sites of backbone cleavage occurred at residues 246 and 259. New findings suggest that cleavage at these sites may be a result of nonenzymatic truncation at asparagine residues. In addition, this approach revealed previously undetected sites of truncation at residues 249, 260, 261, and 262; phosphorylation at Ser 231 and to a lower extent at Ser 229; and racemization/isomerization of l-Asp 243 to d-Asp and d-iso-Asp. The spatial distribution of C-terminally modified AQP0 within the lens indicated an increase in truncation and racemization/isomerization with fiber cell age, whereas the level of Ser 235 phosphorylation increased from the outer to inner cortex but decreased in the nucleus. Furthermore, the remarkably similar pattern and distribution of truncation products from lenses from three donors suggest specific temporal mechanisms for the modification of AQP0.     

Structural models of the supramolecular organization of AQP0 and connexons in junctional microdomains

Membrane proteins perform many essential cellular functions. Over the last years, substantial advances have been made in our understanding of the structure and function of isolated membrane proteins. However, like soluble proteins, many membrane proteins assemble into supramolecular complexes that perform specific functions in specialized membrane domains. Since supramolecular complexes of membrane proteins are difficult to study by conventional approaches, little is known about their composition, organization and assembly. The high signal-to-noise ratio of the images that can be obtained with an atomic force microscope (AFM) makes this instrument a powerful tool to image membrane protein complexes within native membranes. Recently, we have reported high-resolution topographs of junctional microdomains in native eye lens membranes containing two-dimensional (2D) arrays of aquaporin-0 (AQP0) surrounded by connexons. While both proteins are involved in cell adhesion, AQP0 is a specific water channel whereas connexons form cell–cell communication channels with broad substrate specificity. Here, we have performed a detailed analysis of the supramolecular organization of AQP0 tetramers and connexon hexamers in junctional microdomains in the native lens membrane. We present first structural models of these junctional microdomains, which we generated by docking atomic models of AQP0 and connexons into the AFM topographs. The AQP0 2D arrays in the native membrane show the same molecular packing of tetramers seen in highly ordered double-layered 2D crystals obtained through reconstitution of purified AQP0. In contrast, the connexons that surround the AQP0 arrays are only loosely packed. Based on our AFM observations, we propose a mechanism that may explain the supramolecular organization of AQP0 and connexons in junctional domains in native lens membranes.     

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: End truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration

Background

Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions.

Methods

The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2–6 (AQP0-N-del-2-6), AA235–263 (AQP0-1-234), AA239–263 (AQP0-1-238), AA244–263 (AQP0-1-243), AA247–263 (AQP0-1-246), AA250–263 (AQP0-1-249) and AA260–263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays.

Results

AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263).

Conclusions

Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens.

General significance

Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.