SNARE HYPOTHESIS AT NERVE ENDINGS

描述：（研究者摘要）：许多关键蛋白质参与 膜靶向和突触囊泡神经递质释放已经被 确定了一组基本的相互作用，并将其置于 这就是所谓的陷阱假说。 然而，尽管最近取得了迅速进展， 在识别膜靶向和融合的分子组分中， 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很难定义。 是 拟议研究的长期目标是确定和了解 控制SNARE蛋白相互作用的调节机制， 从而调节神经递质和神经激素的释放和突触 可塑性 初步分子/生物化学研究结合 功能研究监测膜电容变化，作为衡量 全细胞膜片钳下的外吞-内吞活性表明， Sec 1家族的蛋白具有重要的调节控制功能。 提出的实验将检验假设，nSec 1蛋白（nSec 1 p） 通过与突触融合蛋白的特异性相互作用调节神经分泌 并且这种相互作用增强了分泌颗粒对接。 拟议 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 允许在200 ℃下解析分泌过程的分子事件， 神经末梢比其他任何神经都要详细 结局 研究人员将利用分子， 生物化学和膜片钳技术来表征调节 nSec 1蛋白相互作用，并分析对Ca 2+的功能影响 电流和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 nSec 1 p上的磷酸化位点，并检查蛋白质的调节 通过特定激酶活性和去极化刺激的相互作用，2） 确定nSec 1 p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)为了确定nSec 1 p对胞吐作用的调节是否利用了 陷阱假说提出的机制和机制，以及4） 确定nSec 1 p是否与cdk 5交互并激活cdk 5， 相互作用受nSec 1 p磷酸化调节，如果存在 对分泌功能的影响。 总而言之， nSec 1 p调节SNARE蛋白相互作用的机制可能是 对充分理解短期和长期的 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.