MODULATION AND REGULATION OF NEURONAL CALCIUM CHANNELS

神经系统通过电合成和传递信息。 化学信使的活动和释放。之间的耦合 电和化学信号传导由钙（Ca）离子流入启动 通过电压门控钙通道。通过一系列鲜为人知的 或完全未知的步骤，钙触发胞吐发射器-或 含囊泡。在过去的两年里， 发生在分离和测序的蛋白质参与 囊泡膜与质膜的融合和回收。几乎 没有人知道它们的功能。拟议的工作将探讨 最近发现的几种蛋白质的功能。 钙偶联分泌的儿茶酚胺和肽类激素将 在大鼠嗜铬细胞和离体神经末梢中研究 膜片钳技术及电容检测 法通过这项技术，我们可以监测到地表的微小变化， 膜面积由胞外或内吞作用产生，同时 控制和测量Ca进入。电容检测技术 具有极好的时间分辨率，报告毫秒范围内的事件， 和灵敏度，区分单个囊泡融合。全细胞 记录模式提供了操纵细胞内的机会 环境，通过引入钙螯合剂，第二信使和蛋白质， 牢房钙水平将通过使用打开的协议来操纵 电压门控Ca通道或通过笼状Ca或笼状Ca- 螯合剂化合物。 我们将开始研究两个小G蛋白Rab 3的性质 和Rab 5，分别参与胞吐和胞吞作用。所有 Ha-ras样G蛋白的Rab亚组的成员与 膜交通的具体步骤。Rab 3的功能与调控 和Rab 5将通过用反义寡核苷酸消除蛋白质来测试。 寡核苷酸，在移液管中急剧引入过量的蛋白质，和 慢性过表达的蛋白质和突变体产生的网站 定向诱变 突触传递机制正迅速被揭示， 比任何人在一两年前所能预测的都要复杂。沿着 随着分泌的基本过程的解开，新的信息是， 揭示了短期和长期调节的巨大潜力 这些过程。拟议中的工作是朝着全面 了解突触传递

The nervous system synthesizes and conveys information by electrical activity and release of chemical messengers. The coupling between electrical and chemical signalling is initiated by calcium (Ca) ion influx through voltage-gated Ca channels. Through a sequence of poorly understood or completely unknown steps, Ca triggers exocytosis of transmitter- or hormone-containing vesicles. In the past two years, explosive progress has occurred in the isolation and sequencing of proteins involved in the fusion and retrieval of vesicle membrane from plasma membrane. Almost nothing is known of their function. The proposed work will probe the function of several of the recently identified proteins. Ca-coupled secretion of catecholamines and peptide hormones will be studied in chromaffin cells and isolated nerve endings of the rat neurohypophysis with patch clamp methods and the capacitance detection technique. With this technique, we can monitor small changes in surface membrane area resulting from exo- or endocytosis, while simultaneously controlling and measuring Ca entry. The capacitance detection technique has both superb temporal resolution, reporting events in the msec range, and sensitivity, discriminating single vesicle fusions. The whole-cell recording mode provides the opportunity to manipulate the intracellular milieu, by introducing Ca chelators, second messengers, and proteins into the cell. Ca levels will be manipulated by using protocols which open voltage-gated Ca channels or by photolysis of caged Ca or caged Ca- chelator compounds. We will begin by investigating the properties of two small G proteins Rab3 and Rab5, implicated in exocytosis and endocytosis, respectively. All members of the Rab sub-group of Ha-ras like G proteins are linked to specific steps of membrane traffic. The function and regulation of Rab3 and Rab5 will be tested by eliminating the proteins by antisense oligonucleotides, acutely introducing excess protein in the pipette, and chronically overexpressing the proteins and mutants created by site directed mutagenesis. Synaptic transmission machinery is rapidly being revealed as much more complex than anyone could have predicted even one or two years ago. Along with unraveling the basic processes of secretion, the new information is revealing an incredibly rich potential for short and long term modulation of these processes. The proposed work represents a basic step toward fully understanding synaptic transmission.