SNARE HYPOTHESIS AT NERVE ENDINGS

神经末梢的圈套假设

• 批准号: 2469187
• 负责人: EDWARD L STUENKEL
• 金额: $ 21.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2469187
• 关键词: calcium flux; enzyme activity; exocytosis; fluorescent dye /probe; hypothalamus; laboratory rat; nerve endings; neural plasticity; neurobiology; neurohypophysis; neurotransmitter transport; phosphorylation; protein kinase; secretion; syntaxin; voltage /patch clamp

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.

描述：（研究者摘要）：许多关键蛋白质参与 膜靶向和突触小泡神经递质释放 确定并定义一组基本的交互，并将其放置在 模型称为 SNARE 假设。 然而，尽管最近进展迅速 识别膜靶向和融合的分子成分 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不明确。 这是 拟议研究的长期目标是识别和理解 控制 SNARE 蛋白相互作用的调控机制， 从而调节神经递质和神经激素的释放以及突触 可塑性。 初步分子/生化研究结合 监测膜电容变化的功能研究，作为衡量 全细胞膜片钳下的胞外胞吞活性表明 Sec1 家族的蛋白质具有重要的调节控制功能。 提出的实验将检验 nSec1 蛋白 (nSec1p) 的假设 通过与突触融合蛋白的特定调节相互作用来调节神经分泌 并且这种相互作用增强了分泌颗粒对接。 拟议的 实验将利用下丘脑-神经垂体的神经末梢 系统，具有独特的解剖学和电生理学优势 允许解决分泌过程的分子事件 神经末梢比任何其他神经都需要更详细的研究 结局。 研究人员将结合分子、 生物化学和膜片钳技术来表征调节 nSec1 蛋白质相互作用并分析对 Ca2+ 的功能影响 电流以及分泌的幅度和动力学（毫秒分辨率） 在个别神经末梢。 具体目标是：1）确定 nSec1p 上的磷酸化位点并检查蛋白质的调节 特定激酶活性和去极化刺激的相互作用，2) 确定 nSec1p 调节分泌颗粒的机制 胞吐作用并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分子 SNARE 假说提出的机制和机制，以及 4) 确定 nSec1p 是否与 cdk5 交互并激活 cdk5，这是否 相互作用受 nSec1p 磷酸化调节，如果存在 对分泌的功能影响。 总而言之，对以下内容的理解 nSec1p 调节 SNARE 蛋白相互作用的机制可能是 对于充分理解短期和长期过程至关重要 突触可塑性和记忆。