The long-term goal of this application is to determine the role of gap junctions in the lens. Gap junctional communication is important for the development and physiology of the lens, as demonstrated by the generation of lens connexin knockout mice. For a better understanding of the physiology of the lens, it is important to study the regulation of gap junctional communication in this tissue. This regulation could occur at the level of assembly, degradation, or channel activity of gap junctions. Our hypothesis is that a PDZ domain-containing protein, ZO-1, acts as an adapter to link other proteins, such as proteases, kinases, and cytoskeletal components, to the lens gap junctions to possibly generate a signaling complex. This complex may regulate the gap junctions directly and/or may be involved in signaling pathways that influence the development and physiology of the lens. Several strategies to study the role of ZO-1 in the function of lens gap junctions are proposed. The first will employ a variation of the yeast two-hybrid screen to identify the proteins (CIP) that interact with the COOH terminal region of lens connexins and that are dependent on ZO-1 for their binding. The second strategy will be to use a lens connexin truncation mutant to disrupt the binding of ZO-l to lens gap junctions. The effect of these alterations will be assessed through the use of a combination of morphological, biochemical, cell biological, and electrophysiological techniques, initially in heterologous expression systems. Subsequently, a mouse will be generated in which the truncated alpha3 connexin replaces the endogenous gene by homologous recombination techniques.
The Specific Aims are designed to test our hypothesis that: 1) ZO-1 in the lens acts as an adapter to link a number of proteins to the lens gap junction, and 2) the interaction of ZO-1 is involved in the regulation of lens gap junctions and is important for lens development and physiology. These studies will enhance our knowledge of the processes and mechanisms that are required for regulation of gap junctions channels in the normal physiology of the lens and during cataractogenesis.
