Regulation of Neuronal Nicotinic Receptors

描述（由申请人提供）： 尼古丁是一种有效的中枢神经系统兴奋剂，其作用是 通过与神经元烟碱受体的相互作用介导， 受体由a或a和13个亚基组成。含a4和132的受体 亚基是大脑中最丰富的受体，并且表现出高的 对激动剂的亲和力。这些受体结合并表现出增加的 密度持续暴露于尼古丁，虽然机制 介导这些效应的机制尚不清楚。基于研究 这表明从大鼠脑中分离的或在大鼠脑中表达的A4132受体， 异种卵母细胞被蛋白激酶磷酸化， 在HEK 293细胞中稳定表达的α 4 B2受体的表达和功能是 被蛋白激酶的激活剂和抑制剂修饰， 增强受体磷酸化，这些受体可能是 受磷酸化/去磷酸化机制调节，翻译后 调节其他配体门控离子通道的过程。这些目标 研究的目的是阐明磷酸化/去磷酸化的作用， 尼古丁长期给药效应的调节机制 或烟碱激动剂。这一目标将通过检验假设来实现 A4132神经元烟碱受体的A4亚单位是 通过特异性蛋白激酶/磷酸酶磷酸化/去磷酸化， A4亚基磷酸化状态的改变， 持续暴露于尼古丁或尼古丁激动剂会改变 受体的为了验证这一假设，将进行细胞和分子研究。 使用表达大鼠α 4 B2受体的非洲爪蟾卵母细胞、SH-EPI细胞 稳定表达人α 4 B2受体的瞬时转染SH-EP-1细胞 用野生型132亚基表达突变型a4亚基， 培养物中，对应于主要胞质结构域的融合蛋白， α 4亚基内的序列和对应于α 4亚基内的序列的合成肽。 a4亚基的胞质结构域，含有假定的磷酸化 网站. 这些研究将确定：I）磷酸化状态的变化 受体的功能; 2）哪些氨基酸是 磷酸化/去磷酸化和哪些酶参与; 3）尼古丁如何 和烟碱激动剂改变磷酸化/去磷酸化的 受体; 4）磷酸化是否改变受体功能。 研究结果将为研究a4132受体的调控提供必要的知识。 了解正常的大脑功能和长期的后果， 暴露于尼古丁和尼古丁激动剂，具有 一直被滥用，并继续被用于治疗。

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.