Regulation of Neuronal Nicotinic Receptors

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DESCRIPTION (provided by applicant): Nicotine is a potent central nervous system stimulant whose effects are mediated through interactions with neuronal nicotinic receptors, a family of receptors composed of a or a and 13 subunits. Receptors containing a4 and 132 subunits are the most abundant receptors in the brain, and exhibit high affinity for agonist. These receptors inactivate and exhibit an increased density following sustained exposure to nicotine, although the mechanisms mediating these effects are not clearly unders aboutod. Based on studies indicating that a4132 receptors either isolated from rat brain or expressed in Xeno pus oocytes are phosphorylated by protein kinases, that the surface expression and function of a4B2 receptors stably expressed in HEK 293 cells are modified by activators and inhibitors of protein kinases, and that nicotine enhances receptor phosphorylation, it is likely that these receptors may be regulated by phosphorylation/ dephosphorylation mechanisms, post-translational processes that regulate other ligand-gated ion channels. The goal of these studies is to elucidate the role of phosphorylation/dephosphorylation mechanisms in mediating the effects of the chronic administration of nicotine or nicotinic agonists. This goal will be achieved by testing the hypothesis that a4 subunits of a4132 neuronal nicotinic receptors are phosphorylated/dephosphorylated by specific protein kinases/phosphatases and that alterations in the phosphorylation state of the a4 subunit following sustained exposure to nicotine or nicotinic agonists alters the function of the receptor. To test this hypothesis, cellular and molecular studies will be carried out using Xenopus oocytes expressing rat a4B2 receptors, SH-EPI cells stably expressing human a4B2 receptors, transiently transfected SH-EP-1 cells expressing mutant a4 subunits with wild-type 132 subunits, primary neuronal cultures, fusion proteins corresponding to the major cytoplasmic domain of the a4 subunit, and synthetic peptides corresponding to sequences within the cytoplasmic domain of the a4 subunit that contain putative phosphorytation sites. These studies will determine: I) whether changes in the phosphorylation state of the receptor affect its function; 2) which amino acids are phosphorylated/dephosphorylated and wnich enzymes are involved; 3) how nicotine and nicotinic agonists alter the phosphorylation/ dephosphorylation of the receptor; and 4) whether phosphorylationidephosphorylation alters receptor function. Results will provide knowledge on the regulation of a4132 receptors which is necessary for understanding both normal brain function and the long-term consequences of exposure to nicotine and nicotinic agonists, pharmacological agents that have been and continue to be abused, as well as used therapeutically.