The lens of the eye is a complex organ that, with the cornea, focuses an image on the retina. Lens cells have glucose transporters, chloride channels, sodium and potassium channels, gap junctions, and many other standard transport proteins in common with the cells of other organs. Aquaporin zero (AQP0) however is a unique water channel protein found only in the fiber cells of the lens. This proposal examines the roles of AQP0 using a combination of a biophysical measure of the water permeability induced by AQP0, molecular biology to design and construct informative mutants of AQP0 and investigation of the effects of adding, altering or deleting AQP0 in the lens of living animals. In this proposal we investigate the role of AQP0 in development and maintenance of the lens by disrupting its normal expression in the zebrafish (which has two AQP0 genes which differ in their properties), either by knocking down expression of AQP0 genes using antisense morpholino oligos or by over expressing variant AQP0 molecules under lens specific promoters (Aim 1) or by combining these techniques. In parallel, we investigate the molecular basis for the regulation of AQP0 water permeability by calcium and hydrogen, two ions whose concentration varies considerably from the edge of the lens to its center by expressing wild type and mutant cRNAs in Xenopus oocytes, then measuring the resulting permeability using a swelling assay. In particular we will examine cooperativity between monomers (Aim 2) and test the hypothesis that the regulation of permeability by calcium is effected by a reversible, calcium- dependent calmodulin block of half of the AQP0 monomers in a tetramer (Aim 3).
Our research aims at understanding the development and aging of the lens of the eye. We believe that such an understanding will lead to an ability to delay or possibly eliminate cataract surgery and its possible complications. Delaying cataract surgery by even a few years or reducing its most common complications could reduce health costs by a very significant amount.
| Fields, James B; Németh-Cahalan, Karin L; Freites, J Alfredo et al. (2017) Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop. J Biol Chem 292:185-195 |
| Reichow, Steve L; Clemens, Daniel M; Freites, J Alfredo et al. (2013) Allosteric mechanism of water-channel gating by Ca2+-calmodulin. Nat Struct Mol Biol 20:1085-92 |
| Clemens, Daniel M; Németh-Cahalan, Karin L; Trinh, Lien et al. (2013) In vivo analysis of aquaporin 0 function in zebrafish: permeability regulation is required for lens transparency. Invest Ophthalmol Vis Sci 54:5136-43 |
| Hall, James E (2012) Through a glass darkly. EMBO Mol Med 4:1-2 |
