PROTEIN DEPHOSPHORYLATION IN NEUROSECRETION

描述(申请人摘要)：蛋白质的作用 钙/钙调蛋白依赖介导的去磷酸化 蛋白磷酸酶(钙调神经磷酸酶)，在调节 哺乳动物神经末梢的神经递质释放 将对大脑进行研究。拟议的研究将提供 洞察突触中钙离子调节的过程， 有助于实现描述 涉及神经分泌的分子机制。 具体地说，生理学和药理学 钙调神经磷酸酶在分离的脑神经末梢中的调节 将会被检查。这些研究将检测钙离子- 外源性和内源性的依赖性去磷酸化 离体脑神经末梢中的蛋白磷酸底物 分数。外源底物去磷酸化将 测量以评估神经中钙调神经磷酸酶的程度 终末可被钙离子和钙调蛋白激活。内源性 依赖于钙离子的去磷酸化底物将是 标准放射性标记后在完整终端中的研究 内源性ATP，集中在两种称为P96的磷酸蛋白上 和p139，已知能显著去磷酸化的蛋白质 去极化诱导钙离子内流。生理调节 其中的去磷酸化事件将由 比较去极化引起的变化的动力学 钙调神经磷酸酶动力学与细胞内钙离子水平的关系 激活，并通过确定 钙离子对去磷酸化至关重要的变化(例如，钙 通道、内质网、Na/Ca逆向转运蛋白)。 将使用各种不同的药物来研究药理调节 已知的改变终端内钙水平的药物(例如 卡巴胆碱)。钙调神经磷酸酶在神经递质中的作用 摄取和释放将由单独的 介绍激活的钙调神经磷酸酶，抑制性合成 多肽(源自钙调神经磷酸酶的调节区)， 和钙调神经磷酸酶抗体进入分离的神经末梢， 使用瞬时冻结/融化渗透法，并检查 神经递质摄取和释放的后续影响。 内源蛋白的纯化及性质研究 将进行底物P96和P139。利用 针对纯化的蛋白和/或 对其氨基酸序列的部分了解，分离 大鼠脑cDNA文库中编码P96和P139的cDNA 将会被尝试。P96和P139在神经中的作用 终端功能将通过单独介绍 这些蛋白质底物和针对这些的抗体 蛋白质进入隔离的末端，并评估随后的 影响神经递质的摄取和释放。因为 钙调神经磷酸酶在大脑中的分布是不同的， 主要分布在纹状体和海马体， 确定钙调神经磷酸酶在神经末梢中的作用可能 提供对中更改的功能的新见解 涉及这些大脑的神经病理疾病状态 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.