SNARE HYPOTHESIS AT NERVE ENDINGS

描述：(研究人员摘要)：许多关键蛋白质参与 膜靶向和突触囊泡神经递质释放 确定并定义一组基本交互，并将其放置在 该模型被称为陷阱假说。然而，尽管最近取得了快速进展， 在鉴定膜靶向和融合的分子组成方面 机器，调节影响促进或抑制蛋白质 相互作用或相互作用的顺序仍然定义不清。它是 拟议研究的长期目标是确定和理解 调控SNARE蛋白相互作用的调控机制(S)， 从而调节神经递质、神经激素释放和突触 可塑性。初步的分子/生化研究结合了 监测膜电容变化的功能研究，作为一种测量 全细胞膜片钳下的胞外吞噬活性，已表明 Sec1家族的蛋白质具有重要的调控功能。 提出的实验将检验nSec1蛋白(NSec1p)的假设 通过与合成素的特定调节相互作用来调节神经分泌 这种相互作用增强了分泌颗粒的对接。建议数 实验将利用下丘脑-神经垂体的神经末梢 系统，具有独特的解剖和电生理优势 允许分解分泌过程的分子事件 对神经末梢的研究比对任何其他神经都要详细 结局。调查人员将利用分子和分子的组合， 生化和膜片钳技术来表征细胞周期调控 NSec1蛋白相互作用及功能对钙离子的影响 电流和分泌物的幅度和动力学的毫秒分辨率 在单独的神经末梢。具体目标是：1)确定 NSec1p的磷酸化位点及其对蛋白质调控的研究 通过特定的激酶活性和去极化刺激的相互作用，2)到 确定nSec1p调节分泌颗粒的机制 胞吐作用和阐明磷酸化状态对分泌的影响， 3)确定nSec1p对胞吐作用的调节是否利用了分子 圈套假说提出的机制和机制，以及4) 确定nSec1p是否与CDK5相互作用并激活，这是否 相互作用由nSec1p磷酸化调节，如果有 对分泌物的功能影响。总而言之，对 NSec1p调节SNARE蛋白相互作用的机制可能是 对于充分理解 突触可塑性和记忆。

DESCRIPTION: (Investigator's Abstract): Many key proteins involved in membrane targeting and synaptic vesicle neurotransmitter release have been identified and a fundamental set of interactions defined and placed in a model termed the SNARE hypothesis. However, despite recent rapid progress in identifying molecular components of the membrane targeting and fusion machine, regulatory influences facilitating or inhibiting the protein interactions or the sequence of interactions remain poorly defined. It is the long range goal of the proposed research to identify and understand the regulatory mechanism(s) which govern SNARE protein interactions and, thereby, regulate neurotransmitter and neurohormone release and synaptic plasticity. Preliminary molecular/biochemical studies combined with functional studies monitoring membrane capacitance changes, as a measure of exo-endocytotic activity under whole cell patch clamp, have indicated that proteins of the Sec1 family serve an important regulatory control function. Experiments proposed will test the hypothesis that nSec1 protein (nSec1p) regulates neurosecretion via a specific regulated interaction with syntaxins and that this interaction enhances secretory granule docking. The proposed experiments will utilize nerve endings of the hypothalamo-neurohypophysial system, which possess unique anatomical and electrophysiological advantages allowing resolution of the molecular events of the secretory process at nerve endings to be studied in greater detail than at any other nerve endings. The investigators will utilize a combination of molecular, biochemical, and patch clamp techniques to characterize the regulation of nSec1 protein interactions and to analyze functional effects on Ca2+ currents and on the amplitude and kinetics of secretion with msec resolution at individual nerve endings. The specific aims are: 1) to determine the sites of phosphorylation on nSec1p and examine regulation of protein interactions by specific kinase activity and depolarizing stimuli, 2) to determine the mechanism by which nSec1p regulates secretory granule exocytosis and elucidate the effects of phosphorylation state on secretion, 3) to determine if nSec1p regulation of exocytosis utilizes the molecular mechanisms and machinery proposed by the SNARE hypothesis, and 4) to determine if nSec1p interacts with and activates cdk5, whether this interaction is regulated by nSec1p phosphorylation, and if there are functional consequences on secretion. In summary, an understanding of the mechanisms by which nSec1p regulates SNARE protein interactions may be essential to full understanding of both short and long-term processes of synaptic plasticity and memory.