Aquaporin-0

Aquaporin-0 (AQP0), formerly known as major intrinsic protein (MIP), is a member of the ubiquitous aquaporin family. In contrast to most mammalian aquaporins, which are expressed in more than one tissue, AQP0 is highly specific to the lens, where it is only expressed in the fiber cells. AQP0 is a rather poor water pore. At neutral pH, AQP0 water permeability is approximately 40 times lower than that of AQP1. AQP0 is however a highly abundant integral membrane protein in lens fiber cells. The vast number of water pores expressed in the plasma membrane would seem to compensate for the rather poor water conductance of individual AQP0 molecules. Furthermore, recent studies have demonstrated that AQP0 water conductance can double under mildly acidic conditions, such as those found in the core of the lens. Unlike all other aquaporins, AQP0 is known to be present in single membranes as well as in membrane junctions between lens fiber cells. It is particularly enriched in the 11-13 nm thin junctions, which are abundant in the more acidic lens core and feature square AQP0 arrays. The propensity of AQP0 to interact with itself has also been demonstrated by reconstitution of the protein into liposomes, which aggregated only when AQP0 was present. When AQP0 was reconstituted into two-dimensional (2D) crystals, electron and atomic force microscopy analysis revealed that these crystals were double-layered with the extracellular surfaces of the AQP0 molecules making specific interactions.

The causes that induce AQP0 to form membrane junctions are yet to be identified. In the differentiation and maturation of lens fiber cells, AQP0 undergoes several post-translational modifications including deamidation and phosphorylation on its carboxyl tail. As fiber cells grow older and become buried more deeply in the lens, some of the AQP0 is C-terminally cleaved at various sites in an age-dependent manner. This process is accelerated in cataract formation. While it has been reported that C-terminal cleavage does not affect AQP0 water conductance across the fiber cell membrane, in collaboration with Joerg Kistler (University of Auckland) we could show that C-terminal truncation enhances the adhesive properties of the extracellular surface of AQP0, resulting in the formation of crystalline junctions in reconstitution experiments.


Purified full-length AQP0 from the lens cortex was reconstituted into artificial bilayers by slow removal of DM in the presence of synthetic lipid (DMPC). At a lipid-to-protein ratio (LPR) of 0.1 (w/w), small vesicles formed containing full-length AQP0. When imaged by negative stain electron microscopy, the vesicles were evenly distributed on the carbon support film (left panel). Reconstitution of core AQP0 using the same conditions yielded vesicles containing both full-length and truncated AQP0. In this case negative stain electron microscopy showed that the resulting vesicles were not evenly dispersed on the carbon film but formed large clusters (right panel).


When full-length AQP0 was reconstituted at an LPR of 0.25, large (~2 µm in diameter) crystalline sheets formed (top left panel). Image processing of glucose-embedded crystals revealed a unit cell with lattice parameters of a = b = 65.5 Å and p4 symmetry. The projection map of the AQP0 tetramer at 4 Å resolution (bottom left panel) is virtually identical to a 3.5 Å projection structure of AQP1, demonstrating that 2D crystals formed by full-length AQP0 isolated from the lens cortex are single-layered. The same reconstitution was performed using AQP0 isolated from the lens core, containing a mixture of full-length and truncated AQP0. In this case large membrane sheets formed (> 6 µm) that in some cases showed two parallel edges, revealing them to be double-layered (top right panel). These double-layered crystals had the same lattice constants as those observed in single-layered crystals reconstituted from cortical full-length AQP0 (a = b = 65.5 Å), but now had a p422 symmetry. A 4 Å projection map calculated from the double-layered crystals showed a tetrameric structure with the same dimensions as an AQP0 tetramer, but each “monomer” now displaying two-fold mirror symmetry (bottom right panel).

» Gonen et al. (2004) J. Mol. Biol. 342:1337-1345.

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The lens-specific AQP0 is the only aquaporin known to form membrane junctions in vivo. Using AQP0 from the core of sheep lenses, where some of the AQP0 is proteolytically cleaved near the C-terminus at various sites in an age-dependent manner, we produced large (> 6 µm) double-layered 2D crystals. The crystals showing p422 symmetry had lattice constants of a = b = 65.5 Å and a thickness of 11 nm, the same dimensions as thin junctions in the lens. Double-layered AQP0 2D crystals are therefore likely to recapitulate thin lens fiber cell junctions. Sequence alignment shows AQP0 to be closely related to the pure water channel AQP1 (43.6% identity, 62.6% similarity). We determined the structure of the AQP0 membrane junction using only electron diffraction patterns. Since the crystal structure of the homologous bovine AQP1 was available, we determined the structure of the AQP0 membrane junction by molecular replacement. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mediated mainly by proline residues conserved in AQP0s from different species but not present in most other aquaporins. While all aquaporin structures determined to date show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.

Electron diffraction patterns of untilted double-layered AQP0 2D crystals typically showed diffraction spots to a resolution better than 3.5 Å (left panel). Electron diffraction patterns of AQP0 crystals were collected at tilt angles up to 70° rather than the usual 60°, thus significantly reducing the extent of the missing cone. Diffraction patterns of AQP0 2D crystals tilted to 70° still showed strong and sharp diffraction spots to 3.5 Å resolution, even in the direction perpendicular to the tilt axis (right panel). The white line represents the orientation of the tilt axis.


Loop C in AQP0, connecting α-helices 3 and 4, is significantly shorter than in AQP1 and GlpF. The shortened loop C is crucial for the formation of the very tight AQP0 junction (left panel), since it allows three specific interactions to be formed that are mediated almost exclusively by proline residues. The most striking interaction involves Pro38, in extracellular loop A. Prolines 38 from all eight symmetry-related AQP0 molecules in the stacked tetramers come together to form a unique rosette-like structure in the very center of the junction (top right panel). The other two interactions involve three AQP0 molecules, which we designated as A, B, and C (bottom right panel). The contacts are formed by residues in the shortened loop C, namely a Pro-Pro motif (Pro109 and Pro110), which is part of a 1-turn helix (helix HC), and residues Arg113 and Pro123.


In top views, the water pore can readily be seen in AQP1 (top left panel), whereas the AQP0 pore appears much more constricted (top right panel). About half of the residues lining the pores differ between AQP0 and AQP1, and many residues in AQP1 are substituted with larger and more hydrophobic ones in AQP0. The AQP1 pore (bottom left panel) has a single constriction site formed by residues Phe58, His182, Arg197, and Cys191. This so-called ar/R site in the pore of junctional AQP0 (bottom right panel) is formed by the corresponding residues Phe48, His172, Arg187, and Ala181, but it shows noteworthy differences. Ala181 replaces AQP1’s Cys191, thus rendering AQP0 water conductance insensitive to mercurials. His172 shifts towards the center of the pore as compared to AQP1’s His182, resulting in a minimum pore diameter of 1.96 Å, which is too narrow for water permeation. While the ar/R constriction site in AQP1 is only ~2 Å long, constriction site I in AQP0 has a length of about 10 Å extending further towards the extracellular surface as well as further into the pore. AQP0 contains a second, novel constriction site in the cytoplasmic half of the pore. The side chain of Tyr149 extends into the water pathway and together with Phe75 and His66 constricts the pore to a minimum diameter of 2.0 Å, again too narrow for water to pass.

» Gonen et al. (2004) Nature 42: 193-197.

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