The survival and maintenance of transparency in the avascular crystalline lens depend upon the healthy functioning of a few surface cells and intercellular communication between all cells in the organ. Intercellular communication is mediated by channels contained within gap junctions. Protein components of lens gap junctions have been identified and molecularly cloned in our laboratory. The overall goal of this project Is to understand the regulation of lens intercellular communication. Gap junctions can be regulated at three levels: (1) protein modification (phosphorylation) and degradation, (2) channel permeability and gating, and (3) gene expression. Studies are proposed that focus on all three components of the regulation of chick lens gap junction proteins, especially connexin56 (Cx56). Protein studies will biochemically characterize the modification of Cx56 by phosphorylation and the contribution of phosphorylation to channel gating and gap junction assembly. The half-life of Cx56 in lens cells and in transfected cell lines will be determined. The process of degradation of Cx56 (and the lens epithelial connexin, Cx43) will be examined.. Physiological studies will assess the molecular permeability and pharmacological regulation of gap junction channels between lens fiber- like cells (in cultured "lentoids") and those between communication- deficient N2A cells stably transfected with Cx56. The Cx56 sequence and site-directed mutant constructs will be expressed in Xenopus oocytes and studied electrophysiologically to localize molecular determinants of its ion permeation and its ability to form non-junctional "hemi"-channels. Gene regulation studies will characterize the sequence and structure of the Cx56 mRNA and gene and will use transient transfection with Cx56 gene/reporter constructs to delineate the Cx56 promoter. These data will have broad implications regarding intercellular coupling in all tissues. Experimental results may also give a better understanding of the role of gap junctions in the maintenance of lens transparency and cataract formation and potential strategies for altering or augmenting lens intercellular communication.