Alok K. Mitra

Three-dimensional organization of a human water channel

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. 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,. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7Å resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an in-plane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted α-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.

The CHIP28 water channel visualized in ice by electron crystallography

Electron crystallography of frozen-hydrated two-dimensional crystals of deglycosylated human erythrocyte CHIP28 reveals an aqueous vestibule in each monomer leading to the water-selective channel that is enclosed by multiple transmembrane α-helices.

Three-dimensional organization of the aquaporin water channel: What can structure tell us about function?

Publisher Summary The entry and exit of water across the lipid bilayer membrane is a fundamental physiological process necessary for maintaining cell homeostasis, which is crucial to the survival of an organism. The chapter discusses several observations that have been suggested in the presence of water-specific channels or pore in some tissues. These were (a) unusually high osmotic water permeability of, for instance, red blood cell membranes and renal proximal tubular epithelium, which is characterized by low Arrhenius activation energy (E a ) and is too rapid to be explained by passive diffusion of water across the bilayer, (b) reversible inhibition of the high water permeability across red blood cells by mercurial reagents, and (c) radiation inactivation studies of renal brush-border membrane vesicles and erythrocytes that indicated that a protein of ∼30kDa is responsible for the high water permeability. Additional evidence in favor of a protein responsible for water transport was provided when, upon injection of heterologous mRNAs from kidney reticulocytes and amphibian bladder into Xenopus oocytes, which is known to have low water permeability, increased water permeability was elicited.

Three-Dimensional Fold of Human AQP1 Water Channel Determined by Electron Cryo-Crystallography of 2-Dimensional Crystals Embedded in Ice

The entry and exit of water across the cell membrane is a fundamental physiological process. The observed high permeability of water in the case of mammalian erythrocytes and renal proximal tubules led to the discovery of water channels (aquaporins, AQP) (Denker et al., 1988; Smith and Agre, 1991). Aquaporins are members of the MIP (major intrinsic protein) superfamily (Gorin et al., 1984) and are found in both eukaryotes and prokaryotes where they serve as channels for rapid dissipation of osmotic gradients across the lipid bilayer (Agre et al., 1995; Verkman et al., 1996). In order to understand the structural elements that constitute and control the water-selective permeability of AQP1, a detailed knowledge of the high-resolution 3-D structure in the bilayer is essential. To this end, 2-D crystals of AQP1 have been used to examine the structure using electron crystallography. Projection maps of AQP1 were determined by electron cryo-crystallography by three groups (Jap and Li, 1995; Mitra et al., 1995; Walz et al., 1995) who used different conditions for 2-D crystallization and specimen preservation for microscopy. Subsequently, a 7A resolution, 3-D density map of AQP1 was determined by us (Cheng et al., 1997) from an analysis of minimal dose images and electron-diffraction patterns recorded from ice-embedded from ice-embedded 2-D crystals. 3-D density maps, at a similar resolution were reported by the other 2 groups (Li et aL9 1997; Walz et al., 1997) which were determined using specimens preserved in trehalose and the tilted projections comprised of images and electron diffraction patterns (Walz et al., 1997) and only images (Li et al.9 1997).

3D reconstruction from electron micrographs of tilted 2D crystal: structure of a human water channel

In order to understand at the atomic level how a biological macromolecule functions, a detailed knowledge of its 3D structure is essential. Unlike soluble proteins, integral membrane proteins are usually recalcitrant to the growth of large, well-ordered 3D crystals, which is necessary for high-resolution x-ray crystallographic analyses. An alternative approach is to grown thin, one molecule thick 2D crystals in lipid bilayers and apply electron crystallography to solve the structures. Lipids surround the membrane protein in such a 2D crystal, which allows for a direct assay of function. Another notable advantage of electron crystallography is that phases can be directly obtained form the images unlike in the case of x-ray where phases must be determined indirectly by methods such as isomorphous replacement etc. The availability of the phase information partially compensates for the lack of data at the highest resolution (typically approximately 3.5A and beyond) because of low-contrast in the images. We briefly review the method of recording high-resolution data from many tilted views of a 2D crystal, merging of phase and amplitudes form images and diffraction patterns respectively and the calculation of a 3D density map. The results from such an analysis applied to the human water channel is discussed in the context of its structure/function relationship.