The ciliary epithelium is the primary site of aqueous human secretion and pressure regulation in the eye, requiring the water transport across a relatively small surface. However, the secretory process which results in aqueous human production is incompletely understood and the identification of precise cellular mechanisms which underlie this process remains to be established. Passive movement of water across cell membranes is driven by osmotic and hydraulic process that arise from the active ion and osmotic solute transports. Rapid movement of water across membranes requires both a suitable driving force and a high membrane water permeability. Recent evidence suggests the presence of a novel family of water channel proteins that regulates water permeability in a variety of tissues, including kidney, red blood cells, lung, and bovine lens. PCR amplification of a partial gene using degenerate oligonucleotide primers corresponding to the conserved sequences among the water channel proteins and subsequent cloning of the complete gene (see preliminary data) which codes for a novel protein highly homologous with other members of the water channel proteins indicates that a new member of this family is present in bovine ciliary epithelium which may regulate water permeability in this tissue. The initial objective of this project is to clone the full length gene for water channel protein and determine its nucleotide sequence from cDNA libraries constructed from human ciliary epithelium as well as from SV40 transformed cell liens of bovine-derived pigmented and human-derived non- pigmented ciliary epithelium. In addition, we proposed to clone the water channel gene from a cDNA library to be constructed from rabbit ciliary epithelium. The full length cDNAs will be sequenced by the dideoxy-chain termination method using universal primers. The long term goals are; (a) to identify the function of the gene product as a water channel protein using an osmotic swelling assay by microinjecting oocytes from Xenopus laevis with mRNA transcribed from the ciliary epithelium gene and by reconstituting the purified gene product in proteoliposomes; (b) to regulate the function of water channel protein using site-specific mutagenesis technique to identify amino acid residues involved in water transport activity; by studying the effect of various drugs that inhibit aqueous humor production on the expression of m RNA levels of cloned gene from ciliary epithelial cell lines; and by identifying specific domains (if any) of this protein involved in the water channel activity; and (c) to determine the tissue distribution of mRNA encoding water channel proteins from ciliary epithelium by Northern blotting and in situ hybridization; and to localize the expression site and cellular specificity of these proteins by Western blotting and immunocyte/histochemistry using antibodies raised against the purified gene product so as to define their subcellular organization. Identification and characterization of a novel water channel protein in bovine and human ciliary epithelium will provide new insights into specific mechanism(s) which underlie aqueous human transport.
