ROLE OF PROTEIN DEPHOSPHORYLATION IN NEUROSECRETION

描述（申请人摘要）：蛋白质的作用 Ca 2 +/钙调素依赖性介导的去磷酸化 蛋白磷酸酶（钙调神经磷酸酶），在调节 哺乳动物神经末梢释放神经递质 大脑将被研究。 拟议的研究将提供 深入了解突触处的Ca 2+调节过程， 有助于实现描述 参与神经分泌的分子机制。 具体而言，生理和药理学 钙调神经磷酸酶在离体脑神经末梢中的调节 将被审查。 这些研究将检查Ca 2 +- 外源性和内源性依赖性去磷酸化 离体脑神经末梢蛋白磷酸化底物 分数 外源底物去磷酸化将是 测量以评估神经中钙调磷酸酶 终末可被Ca 2+和钙调蛋白激活。 Ca 2+依赖性去磷酸化的底物将 标准放射性标记后，在完整末端中研究 内源性ATP，集中于两种称为P96的磷蛋白 和P139，已知蛋白质在 去极化诱导的Ca 2+内流。 生理调节 这些去磷酸化事件将被探索， 比较去极化诱导的变化的动力学， 终末内Ca ~（2+）水平与钙调神经磷酸酶动力学 激活，并通过确定的性质和来源， Ca 2+变化对去磷酸化至关重要（例如，CA 通道、内质网、Na/Ca反向转运蛋白）。 药理学调节将使用多种方法进行研究， 已知改变末端内Ca 2+水平的药物（例如， 卡巴胆碱）。 钙调神经磷酸酶在神经递质中的作用 吸收和释放将分别由 引入活化的钙调磷酸酶，抑制性合成 肽（衍生自钙调磷酸酶的调节区）， 和钙调神经磷酸酶的抗体进入分离的神经末梢， 使用瞬时冻融渗透，并检查 神经递质摄取和释放的后续效应。 内源蛋白的纯化和鉴定 将采用P96和P139基板。 利用 针对纯化蛋白质产生的抗体和/或 部分了解其氨基酸序列，分离 从大鼠脑cDNA文库中获得编码P96和P139的cDNA 将尝试。 P96和P139在神经系统中的作用 终端功能将通过单独介绍 这些蛋白质底物和针对它们的抗体 蛋白质到分离的终端，并评估随后的 影响神经递质的摄取和释放。因为 钙调神经磷酸酶在整个脑中有差异地分布， 主要存在于纹状体和海马体中， 确定钙调神经磷酸酶在神经末梢中的作用， 提供了新的见解，改变功能， 涉及这些大脑的神经病理学疾病状态 地区

DESCRIPTION (Applicant's Abstract): The role of protein dephosphorylation as mediated by Ca2+/calmodulin-dependent protein phosphatase (calcineurin), in the regulation of neurotransmitter release from nerve terminals in mammalian brain will be studied. The proposed studies will offer insight into Ca2+-regulated processes at the synapse, contributing to the long-term objective of describing the molecular mechanisms involved in neurosecretion. Specifically, the physiological and pharmacological regulation of calcineurin in isolated brain nerve terminals will be examined. These studies will examine the Ca2+- dependent dephosphorylation of exogenous and endogenous protein phosphosubstrates in isolated brain nerve terminal fractions. Exogenous substrate dephosphorylation will be measured to assess the extent to which calcineurin in nerve terminals can be activated by Ca2+ and calmodulin.Endogenous substrates for Ca2+-dependent dephosphorylation will be studied in intact terminals after standard radiolabeling of endogenous ATP, focusing on two phosphoproteins termed P96 and P139, proteins known to dramatically dephosphorylate upon depolarization-induced Ca2+ entry. Physiological regulation of these dephosphorylation events will be explored by comparing the kinetics of depolarization-induced changes in intraterminal Ca2+ levels with the kinetics of calcineurin activation, and by determining the nature and source of the Ca2+ changes essential for dephosphorylation (e.g., Ca channels, endoplasmic reticulum, Na/Ca antiporter). Pharmacological regulation will be studied using a variety of agents known to change intraterminal Ca2+ levels (e.g. carbachol). The function of calcineurin in neurotransmitter uptake and release will be investigated by separately introducing activated calcineurin, inhibitory synthetic peptides (derived from the regulatory region of calcineurin), and antibodies to calcineurin into isolated nerve terminals, using transient freeze/thaw permeabilization, and examining subsequent effects of neurotransmitter uptake and release. Purification and characterization of the endogenous protein substrates P96 and P139 will be undertaken. Utilizing antibodies raised against the purified proteins and/or partial knowledge of their amino acid sequences, isolation of the cDNAs encoding P96 and P139 from rat brain cDNA libraries will be attempted. The roles that P96 and P139 play in nerve terminal function will be addressed by separately introducing these protein substrates and antibodies against these proteins into isolated terminals, and assessing subsequent effects on neurotransmitter uptake and release.Because calcineurin is differentially distributed throughout brain, being predominantly in corpus striatum and hippocampus, determining the role of calcineurin in nerve terminals may provide new insights into altered function in neuropathological disease states involving these brain regions.