In mammalian tissues water transport is of critical importance for osmotic balance and is facilitated by aquaporins (AQPs), which are integral membrane proteins forming aqueous pores. The existence of 9 identified homologous aquaporins with diversive and complex tissue distributions, distinct biophysical, biochemical, physiological and morphological properties, appeals to novel insights of water and solute transport, which could lead to alternative treatments of hypertension, congestive heart failure, cirrhosis, the nephrotic syndrome, and other edema forming states. This proposal addresses a detailed analysis of the molecular physiology of solvent/solute transport by functional domain exchange between water selective AQP4 channels and poly-selective AQP9 channels in combination with a scanning site-directed mutagenesis program. The production of active chimeras (specific aim 1) with mixed functional (selectivity for water and solute, sensitivity to phloretin and HgCI2) and structural (formation of orthogonal array aggregates or tetramers) properties will be studied in chimera-expressing oocytes. By osmotic volume flow and tracer studies water and solute transport will be measured. Freeze fracture in combination with immunogold surface labeling will be utilized to investigate morphology. A second focus will be the purification of over-expressed AQP9 and mutant AQP9 proteins in baculovirus infected insect cells (specific aim 2) for the establishment of the molecular organization of AQP9 by 2-dimensional (2- D) crystallography. Antibodies raised against a synthetic AQP9-C- terminal peptide will be utilized as a marker in SDS gels and Western blots analyses. The procedures for isolation include stripping and extraction by detergents, chromatographic maneuvers, and are similar to those used to purify AQP4. Because purified functional AQP4 is already available, low resolution 2-D crystallography of AQP4 will commence first and will be carried out as has been done for AQP1. The prospect of detailed structural data of AQP4 and AQP9 is attractive and will lead to a better understanding of pore structure and permselectivity, diversity and distinct roles of aquaporins, and may also facilitate the design of water channel diuretics or """"""""aquaretic"""""""" agents that could induce a diuresis in situations where present diuretics, such as furosemide, are ineffective.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine B Study Section (GMB)
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Scherbenske, M James
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Massachusetts General Hospital
United States
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