The long-term objectives of this study are to identify the mechanisms that regulate electrical coupling in the retina, and to determine which modes of regulation are most important for the adaptive processes observed n different electrically coupled neural circuits. Electrical coupling, mediated by gap junctions, contributes to the signal processing functions of most types of retinal neurons. Modulation of gap junctions during visual adaptation has profound effects on sensitivity and receptive field properties of many neurons and influences the path of signal flow in the mammalian rod circuit. In different circuits within the retina, very different demands are placed on gap junctions made of the same connexin proteins, and the regulatory responses observed are quite disparate. Previous results suggest that there are several different mechanisms that regulate coupling, and we hypothesize that the contributions of these will differ depending on the cell type. This project focuses on delineating the major mechanisms that regulate gap junctions made of Cx35/36, the most widespread connexin in retinal neurons and throughout the central nervous system. The specific hypotheses to be examined in this project are that: 1) the phosphorylation state of specific residues characterizes the regulatory state of Cx35 gap junctions; 2) calmodulin is involved in calcium-dependent regulation of gap junction coupling; and 3) regulation of coupling is influenced by proteins closely associated with Cx35 in a complex. A variety of in vitro biophysical and biochemical techniques will be used to characterize posttranslational modifications and protein binding interactions of Cx35. Mutations that disrupt these processes will be made and their effects on regulation studied in cell culture expression systems. The association of components of the regulatory pathways with Cx35 in retinal neurons will be examined by immunostaining. These studies will identify the major sites that could be subject to improper regulation in retinal disorders that affect gap junction coupling. ? ?
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