The long-term goal of this research is to define the critical physiologic changes in the early stages of lens cataract formation. Regulation of connexin-mediated gap junctional communication, and intracellular cytosolic Ca 2+ concentration ([Ca2+]i) are essential for maintaining transparency of the lens of the eye. It has now been demonstrated that the purinergic agonist ATP effects a delayed, transient inhibition, while elevated [Ca2+]i inhibits cell-to-cell coupling mediated by lens gap junctions. The immediate objectives of this project are to define the mechanisms by which ATP and [Ca2+]i regulate lens gap junctions. This will be achieved using a sheep lens cell culture system, as well as HeLa cell lines stably transfected with different lens connexins. These objectives will be achieved in two specific aims.
The first aim will determine the mechanism by which ATP effects the inhibition of lens gap junctions. The hypothesis to be tested in this proposal is that the ATP-dependent, delayed, transient inhibition of lens gap junctions is effected by the protein kinase-catalyzed phosphorylation of the lens connexins. The phosphorylated connexin43 (Cx43) amino acid(s) will be identified, and it will then be demonstrated that the phosphorylation of the amino acid(s) is required for this agonist dependent effect; the delayed, transient nature of this ATP-dependent inhibition will also be defined. Finally, the mechanism by which ATP mediates the delayed, transient inhibition of lens Cx44 will be determined.
The second aim will determine the mechanism by which elevated intracellular Ca 2+ concentration inhibits lens gap junctions. The hypothesis to be tested is that calmodulin (CAM) mediates the Ca2+-dependent inhibition of lens gap junctions via its direct interaction with the lens connexins. The peptide sequence that CaM interacts with in Cx43 to effect this inhibition will be identified and confirmed by mutating these identified CaM binding sequences(s). Finally, it will be determined whether Ca2+-CaM regulates cell-to-cell communication mediated by Cx44. Altered regulation of lens gap junctions and [Ca2+]i are recognized as key factors in ? cataract development. These studies, by defining novel physiologic roles for gap junctions in maintaining lens homeostasis, will allow us to generate new models for cataract formation, and enable the development of new therapies to manage and/or prevent human maturity onset cataract. ? ?
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| Zou, Juan; Salarian, Mani; Chen, Yanyi et al. (2014) Gap junction regulation by calmodulin. FEBS Lett 588:1430-8 |
| Xu, Qin; Kopp, Richard F; Chen, Yanyi et al. (2012) Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain. Am J Physiol Cell Physiol 302:C1548-56 |
| Chen, Yanyi; Zhou, Yubin; Lin, Xianming et al. (2011) Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin. Biochem J 435:711-22 |
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| Zhou, Yubin; Yang, Wei; Lurtz, Monica M et al. (2009) Calmodulin mediates the Ca2+-dependent regulation of Cx44 gap junctions. Biophys J 96:2832-48 |
| Lurtz, Monica M; Louis, Charles F (2007) Intracellular calcium regulation of connexin43. Am J Physiol Cell Physiol 293:C1806-13 |
| Zhou, Yubin; Yang, Wei; Lurtz, Monica M et al. (2007) Identification of the calmodulin binding domain of connexin 43. J Biol Chem 282:35005-17 |
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