Structure of aquaporins

The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells. The three-dimensional structure of deglycosylated, human erythrocyte AQP1, was determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer (Cheng, 1997).

Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000 have identified a mercury-sensitive water channel as aquaporin-1 (AQP1). AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. The three-dimensional structure of AQP1 was determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices that form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane (Walz, 1997).

The functions of major intrinsic protein (MIP) in the vertebrate lens of the eye are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibit small, equivalent membrane currents that can be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes is increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increases more than 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins reveal that the 4-kDa cytoplasmic domain of MIP does not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raises the Pf by 50%. Thus oocytes expressing MIP fail to exhibit ion channel activity and consistently exhibit water transport by a facilitated pathway that is qualitatively similar to the aquaporins but of lesser magnitude. It is concluded that MIP functions as an aquaporin in the vertebrate lens, but the protein may also have other essential functions (Mulders, 1995).

Evolutionary and biological diversity of aquaporins

The major intrinsic protein (MIP) of the bovine lens fiber cell membrane was the first member of the MIP family of proteins to be sequenced and characterized. It is probably a homotetramer with transmembrane channel activity that plays a role in lens biogenesis or maintenance. The polypeptide chain of each subunit may span the membrane six times, and both the N- and C-termini face the cell cytoplasm. Eighteen sequenced or partially sequenced proteins from bacteria, yeast, plants, and animals have now been shown to be members of the MIP family. These proteins appear to function in (1) metazoan development and neurogenesis (MIP and BIB), (2) water transport across the human erythrocyte membrane (ChIP), (3) communication between host plant cells and symbiotic nitrogen-fixing bacteria (NOD), (4) transport across the tonoplast membrane during plant seed development (alpha-TIP), (5) water stress-induced resistance to desiccation in plants (Wsi-TIP), (6) suppression of a genetic growth defect on fermentable sugars in yeast (FPS1), and (7) transport of glycerol across bacterial cell membranes (GlpF). One other sequenced member of the MIP family (ORF1 of Lactococcus lactis) has no known physiological function. The biochemical functions of the eukaryotic proteins are not well established. Computer analyses have revealed that the first and second halves of all MIP family proteins probably arose by a tandem, intragenic, duplication event. Thus, the primary structure of putative transmembrane helices 1 to 3 is similar to that of putative transmembrane helices 4 to 6 even though they are of opposite orientation in the membrane. Among the most conserved residues in these two repeated halves are a membrane-embedded glutamate (E) in helices 1 and 4, an asparagine-proline-alanine (NPA) sequence in the loops between helices 2 and 3 (cytoplasmically localized) and helices 5 and 6 (extracellularly localized), and a glycine within helices 3 and 6. Statistical analyses suggest that the two halves of these proteins have evolved to serve distinct functions: the first half is more important for the generalized (that is, common) functions of these proteins, while the second half is more differentiated to provide for specific (more varied or dissimilar) functions. The apparent origin of MIP family proteins by duplication of a three-spanner precursor protein suggests an evolutionary origin distinct from other transport proteins with six transmembrane spanners (Reizer, 1993).

The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity for water and/or neutral solutes. 84 fully or partially sequenced members of this family have beed identified; over 50 representative, divergent, fully sequenced members have been aligned; the resultant multiple alignment has been used to derive current MIP family-specific signature sequences and a phylogenetic tree has been constructed. The tree reveals novel features relevant to the evolutionary history of this protein family. These features plus an evaluation of functional studies lead to two postulates: (1) that all current MIP family proteins derived from two divergent bacterial paralogues, one a glycerol facilitator, the other an aquaporin, and (2) that most or all current members of the family have retained these or closely related physiological functions (Park, 1996).

A group of transmembrane proteins that are related to the major intrinsic protein of lens fibers (MIP26) have been named "aquaporins" to reflect their role as water channels. These proteins are located at strategic membrane sites in a variety of epithelia, most of which have well-defined physiological functions in fluid absorption or secretion. However, some aquaporins have been localized in cell types where their role is at present unknown. Most of the aquaporins are delivered to the plasma membrane in a non-regulated (constitutive) fashion, but AQP2 enters the regulated exocytotic pathway and its membrane expression is controlled by the action of the antidiuretic hormone, vasopressin. These pathways of constitutive versus regulated delivery to the plasma membrane have been reconstituted in transfected LLC-PK1 epithelial cells, indicating that the information encoded within the protein sequence is sufficient to allow sorting of newly synthesized protein into distinct intracellular vesicles. Different members of the aquaporin family can be targeted to either apical, basolateral or both apical and basolateral plasma membrane domains of polarized epithelial cells. This implies that signals for the polarized targeting of these proteins also is located in non-homologous regions of these similar proteins. Thus, future investigations on the aquaporin family of proteins will provide important information not only on the physiology of membrane transport processes in many cell types, but also on the targeting and trafficking signals that allow proteins to enter distinct intracellular vesicular pathways in epithelial cells (Brown, 1995).

A comparison was made of single channel water and glycerol permeabilities of mammalian aquaporins (AQP 1-5) and the major intrinsic protein of lens fiber (MIP). Each of 30 epitope-tagged constructs were expressed strongly at the oocyte plasma membrane. Glycerol was increased significantly in oocytes expressing AQP3 (GLIP). Glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. The different aquaporins have very different intrinsic water permeabilities for the mammalian aquaporin homologs, with the permiability for AQP4 remarkably higher than that of the others. The permiability values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins (Yang, 1997).

GlpF is the glycerol facilitator of the E. coli inner membrane with sequence similarity to Bib. It is a passive channel permeable to small neutral molecules including glycerol and glycine. Although the E. coli outer membrane has several pore-type transporters, glpF is the only channel that allows bidirectional transport of molecules through the inner cytoplasmic membrane (Rao, 1990 and references).

Aquaporins of the eye lens

The major intrinsic protein (MIP) of the vertebrate eye lens is the first identified member of a sequence-related family of cell-membrane proteins that appears to have evolved by gene duplication. Several members of the MIP family transport water (aquaporins), glycerol and other small molecules in microbial, plant and animal cells. Mutations in two aquaporin homologs of MIP underlie an autosomal recessive form of nephrogenic diabetes insipidus and absence of the Colton blood group antigens in humans. Distinct mutations in the murine Mip gene underlie one form of autosomal dominant cataract in the mouse. The cataract Fraser mutation is a transposon-induced splicing error that substitutes a long terminal repeat sequence for the carboxy-terminus of MIP. The lens opacity mutation is an amino-acid substitution that inhibits targeting of MIP to the cell-membrane. These allelic cataract mutations provide the first direct evidence that MIP plays a crucial role in the development of a transparent eye lens (Shiels, 1995).

Major intrinsic protein is the most abundant membrane protein in mammalian lens fiber cells. It is localized on the cell membrane. As the lens is avascular, cell-cell coupling is essential to ensure extensive metabolite exchange. There are abundant lens junctional structures between the fiber cells, some of which resemble gap junctions. It has been postulated that MIP is involved in forming gap junctions between the lens fiber cells. Unlike connexins, however, MIP has not been shown to form gap junctions and MIP is localized not only to gap junctions but also to other types of junctions. MIP has been reconstituted into lipid bilayers and has been shown to form non-selective channels with large conductance (Rao, 1990 and references).

Aquaporins of Malpighian tubules, kidney, and the digestive system