The long term objective of this research is to understand the functional mechanism of water transport across membrane channels. The aquaporins (AQP) are a family of water channel proteins found in bacteria, yeast, insect, plant, mammalian and amphibian tissues and belong to the MIP (Major Intrinsic Protein) superfamily. Aquaporins are critical for the active regulation of water balance required for normal cell function; defects in AQP2, for example, have been related to diseases such as nephrogenic diabetes insipidus. AQP1 is a subset of the aquaporins and can be found to exist in a variety of tissues from organs such as the kidney, gall bladder, spleen, lung, intestine, inner ear and eyes. These channels are believed to be water specific, transporting water across a number of epithelial and endothelial cell layers during fluid absorption and secretion. We propose to continue our efforts in determining the atomic model of AQP1. In the current grant years, we have obtained the projection map of AQP1 at a resolution of about 3.5 Angstrom units resolution and a 3D map at approximately 6 Angstrom units. Concentrated efforts to obtain the 3D structure at 3.5 Angstrom units using electron crystallography were faced with several technical problems: images and diffraction patterns of highly tilted samples show very limited structural information normal to the tilt axis. In addition, the current resolution of structures determined using electron crystallography is not adequate to clearly observe water molecules which is crucial for understanding the aquaporin functional mechanisms and their specificity for water. However, the great difficulty in obtaining 3D crystals of membrane proteins for x-ray crystallographic studies has been a major stumbling block. In the last year, we have focused our efforts on 3D crystallization and were successful in obtaining crystals suitable for x-ray crystallographic studies. Native date sets have been collected to approximately 3 Angstrom units resolution, although the crystals show diffraction spots to better than 2.5 Angstrom units resolution. We are now focusing our major efforts on obtaining heavy atom derivatives. The structure of the water channel at approximately 3 Angstrom units resolution or higher would provide a paradigm for understanding the regulation of the transport of water across the membrane by the MIP superfamily. The atomic model will reveal the molecular details of the channel including the mechanism that regulates its specificity for water transport. It will also provide a structural rationale for the change in specificity, from water to glycerol and the alteration of the protein oligomeric state, from tetramer to monomeric form, observed in a double point mutant of AQPcic. The knowledge of the molecular mechanisms of water channels could provide insights into the structural basis of protein defects resulting in diseases such as nephrogenic diabetes insipidus.
| Han, Bong-Gyoon; Guliaev, Anton B; Walian, Peter J et al. (2006) Water transport in AQP0 aquaporin: molecular dynamics studies. J Mol Biol 360:285-96 |
| Li, H L; Sui, H X; Ghanshani, S et al. (1998) Two-dimensional crystallization and projection structure of KcsA potassium channel. J Mol Biol 282:211-6 |
