Mutations in Cx46 (GJA3) and Cx50 (GJA8) cause cataracts in humans, and mice with targeted deletions of Cx46 and Cx50 also develop cataracts and microphthalmia, providing animals models for the human disease. This clearly shows that the lens needs gap junction channels, but does not address the types of molecules must pass through them to support normal lens growth and clarity. The dual whole cell patch clamp technique allows simultaneous determination of electrical connectivity (junctional conductance) and junctional flux for molecules such as second messengers introduced from the pipette of one cell. We have previously shown that knockout of Cx50, but not Cx43 or Cx46, results in smaller lenses due to a transient decrease in postnatal epithelial cell mitosis. This phenotype cannot be corrected by genetic substitution with Cx46, suggesting that loss of a single connexin species within a given tissue not only changes the total levels of communication, but also alters the kind of molecules being exchanged between the coupled cells. We hypothesize that homotypic Cx43, Cx46 and Cx50 channels will exhibit intrinsic differences in permeability to second messenger molecules like cAMP, IP3 and Ca . We further hypothesize that differences in permeability to second messengers are 2+ critical to the distinct functional roles of Cx43, Cx46 and Cx50 in vivo. We will determine the second messenger permeability of gap junction channels made from Cx43, Cx46 and Cx50 to Ca2+, inositol triphosphate (IP3) and cyclic nucleotides (cAMP). We will also map developmental differences in second messenger permeability through gap junction channels in genetically engineered mouse lenses. These studies seek to define how the diversity in connexin protein subunits influences intercellular communication in the lens, by contrasting the permeability of lens gap junction channels to second messenger molecules. These proposed studies have the potential to improve our understanding of the role of connexin channel permselectivity in lens development, and also as a potential contributor to disease states such as cataract.
The World Health Organization identified cataract as the leading cause of blindness. In the US, cataract extraction is the most common Medicare surgery, adding a financial burden to healthcare of more than $6.8 billion annually. Insights into delaying the onset of age-related cataract and the need for surgery could save billions in healthcare expenditures. Better knowledge of the biological mechanisms that regulate lens intercellular communication will suggest new therapeutic strategies to prevent or postpone the progression of cataract, thereby alleviating the need for surgical intervention.
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