CAMP-DEPENDENT PROTEIN KINASES IN NEURONAL MODULATION

CAMP依赖的蛋白激酶(PKA)是细胞内 在许多神经性疾病中涉及的信号通路 感觉运动性突触的可塑性，包括突触前易化 在海兔中，一种简单形式的学习和记忆的细胞关联。 这种酶传递由神经调节递质携带的信号， 作为动物经历的结果，它们被释放到现场 在靶神经元内改变它们的属性和它们的相互作用 与其他神经元的关系。PKA涉及几个方面的便利化 它们在空间和时间上是分开的。例如，在短时间内- 长期易化，PKA介导钾通道的关闭， 延长动作电位，导致递质增加 放手。在长期易化中，突触生长被激活并 需要PKA对转录因子进行磷酸化。怎么能 一种单一的酶可以控制如此多样化的活动，而不会造成混乱 通过不分青红皂白的磷酸化？许多人的答案在于 海兔PKA的复杂性，它由至少五个调控因子组成 (R)和四个催化(C)亚基，产生多种全酶 (R2C2)。该实验室最近的研究表明，这些形式的PKA 在底物专一性、调节和亚细胞定位方面不同。 我们的假设是，这解释了生理上的多样性 通过提供具有多种生理作用的酶，两者 重叠的和不同的。为了确立这一思想， Aplysia神经元性PKA的各种R和C亚基将被同时检测 在体外和在完整的感觉神经元和细胞提取物中，重点是 关于不同形式的亚基之间的行为差异。 在细胞研究中，诱导短缩和短缩的处理效果 将确定长期便利化。以下问题将 应解决：1、R和C的多样性的性质是什么 海兔感觉神经元中的PKA亚单位？目前的cDNA编码 未知的亚基将被克隆和测序。2.什么是 C亚基的底物？底物专一性将在 在体外，在感觉神经元和匀浆中，以及通过电生理 正在录音。3.海兔神经元中的PKA是如何调控的？特例 将关注R亚基和R亚基的调节 “自动磷酸化”以及其他酶的磷酸化。4. 海兔神经元中的PKA亚单位在哪里，它们会改变位置吗？ 不同形式的R和C亚基的亚细胞位置将 在诱导促进的治疗前后，通过以下方式确定 使用亚细胞分离，免疫荧光显微镜和 荧光成像。中揭示的一般原则可能 这些研究将适用于荧光成像。很有可能 这些研究揭示的一般原则将适用于 脊椎动物。因此，这项工作有助于更好地理解 正常大脑和疾病大脑的可塑性。

cAMP-dependent protein kinase (PKA) is a component of an intracellular signalling pathway that is implicated in many examples of neural plasticity including presynaptic facilitation of the sensorimotor synapse in Aplysia, a cellular correlate of a simple form of learning and memory. The enzyme transduces signals carried by neuromodulatory transmitters, which are released as the result of an animal's experience, to sites within target neurons altering their properties and their interactions with other neurons. PKA is involved in several aspects of facilitation that are spatially and temporally separated. For example, during short- term facilitation, PKA mediates the closure of potassium channels, prolonging the action potential, which results in increased transmitter release. In long-term facilitation, synaptic growth is activated and requires the phosphorylation of transcription factors by PKA. How can a single enzyme control such diverse events, without causing chaos through indiscriminate phosphorylation? The answer many lie in the complexity of Aplysia PKA, which is composed of at least five regulatory (R) and four catalytic (C) subunits that generate multiple holoenzymes (R2C2). Recent work from this laboratory shows that these forms of PKA differ in substrate specificity, regulation, and subcellular location. Our hypothesis is that this accounts for the physiological versatility of PKA, by providing enzymes with multiple physiological roles, both overlapping and distinct. To establish this idea, the properties of the various R and C subunits of Aplysia neuronal PKA will be examined both in vitro and in intact sensory neurons and cell extracts, with emphasis on differences in behavior between the various forms of the subunits. In the cellular studies, the effects of treatments that induce short- and long-term facilitation will be determined. The following questions will be addressed: 1, What is the nature of the diversity of the R and C subunits of PKA in Aplysia sensory neurons? cDNAs encoding presently uncharacterized subunits will be cloned and sequenced. 2. What are the substrates of the C subunits? Substrate specificity will be examined in vitro, in sensory neurons and homogenates, and by electrophysiological recording. 3. How is PKA regulated in Aplysia neurons? Particular attention will be given to regulation by R subunits and by "autophosphorylation" as well as phosphorylation by other kinases. 4. Where are PKA subunits in Aplysia neurons and do they change location? The subcellular locations of individual forms of R and C subunits will be determined, before and after treatments that induce facilitation, by using subcellular fractionation, immunofluorescence microscopy and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable to vertebrates. Thus, this work lead to a better understanding of plasticity in normal and disease brain.