Aquaporins a ubiquitous multigene family expressed throughout the plant and animal kingdom. Currently six aquaporins are recognized; these typically display restricted tissue distributions; their absence may account for a variety of subtle and not-so-subtle functional abnormalities, such as diabetes insipidus or cerebral edema, and even potentially a hereditary form a ataxia (in mice). The current application seeks funding to continue the studies began under the auspices of the initial award. Specifically, molecular experiments are proposed to characterize AQP4 (brain), AQP5 (salivary glands and lung), and newly identified microbial aquaporins.
Four AIMS are outlined: 1) Genetic studies to fully elucidate the alternative MRNA splicing patterns of AQP4, define the genomic organizations of this gene and that of other aquaporins; and develop transgenic models that will elucidate the phenotypic consequences of defective aquaporin function. 2) Tissue distribution studies of AQP4 and AQP5 to explore the expression patterns, developmental appearance, and the factors that alter the expression of these tow isoforms, using both in situ hybridization and immunohistochemistry, as well as promoter-reporter constructs in cell culture. 3) Structural and functional studies on whole proteins (native and mutant aquaporins) to evaluate the subunit organization, post-translational modification, and intersubunit oligomerization of AQP4 and AQP5. These latter experiments will include the expression of AQP4/5 in oocytes, expression in yeast and membrane reconstitution for high-resolution Em, and other biochemical and biophysical characterizations. And finally, 4) Phenotypic investigations to discern the role of the aquaporins in disease states, including the evaluation of rodents with spontaneous mutations or gene disruptions, and the screening of human patients with selected clinical disorders that show promise of being related to defeats in aquaporin function. Putative conditions to be examined include mice with hereditary ataxia and patients with increased intracranial pressure
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