STUDIES OF EXOCYTOSIS AND ENDOCYTOSIS

胞吐作用和内吞作用的研究

• 批准号: 6451504
• 负责人: AARON P. FOX
• 金额: $ 15.94万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6451504
• 关键词: calcium channel; calcium flux; cholinergic receptors; chromaffin cells; dielectric property; exocytosis; muscarinic receptor; neural transmission; neurotransmitter transport; nicotinic receptors; phosphatase inhibitor; phosphorylation; receptor mediated endocytosis; secretion; synapses; voltage /patch clamp; voltage gated channel

This application focusses on mechanisms of exocytosis and endocytosis in chromaffin cells. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. Ongoing studies will verify (or disprove) this hypothesis. In all of our experiments to date, catecholamine release is followed by rapid membrane retrieval (tau of seconds). We will investigate whether membrane retrieval rates are similar for large and small levels of secretion, whether membrane retrieval is Ca2+ dependent, and whether known kinases or phosphatases are important in this process. As we are one of the few labs that measuring retrieval rates in real-time, we believe that we have a contribution to make in understanding this important process. Acetylcholine, released from splanchnic nerve terminals within the adrenal medulla, depolarizes chromaffin cells by activating Ca2+- permeable nicotinic ACh receptors, leading to calcium entry. ACh also promotes calcium release from intracellular stores by activating muscarinic receptors. Three different Ca2+-signals summate to trigger release; these are calcium-entry through Ca2+ channels, calcium-entry through Ca2+-permeable nicotinic ACh receptors and calcium-release from intracellular stores following muscarinic ACh receptor activation. In collaboration with Dr. Green, we will investigate the relative importance of each of these signals, as well as how they work together. N-type Ca channels have been found in virtually every neuron. They are known to be involved in neurotransmitter release at a variety of synapses (as well as chromaffin cells), yet detailed biophysical information has been surprisingly difficult to obtain, in part because N-type Ca channels are found in cells with other types of channels. We have available cloned N-type Ca channels stably expressed in HEK cells. In collaboration with Dr. Richard Miller's lab we plan on carrying out extensive studies of N channel gating currents. This grant application may look diffuse but Specific Aims 3 and 4 are collaborative efforts that strengthen the interactions between my lab and those of Drs. Green and Miller and so should be judged in that context.

本申请集中于细胞内的胞吐和胞吞作用的机制。 嗜铬细胞分泌是由进入细胞的Ca 2+触发的 通过电压门控钙通道。嗜铬细胞有三个 不同类型的钙通道。一种是易化钙通道， 不是由单一的短暂去极化激活的。招聘这些 通道可能涉及一种新形式的通道调节，电压- 依赖性磷酸化正在进行的研究将验证（或反驳）这一点 假说. 在我们迄今为止的所有实验中，儿茶酚胺释放之后是 快速膜修复（τ秒）。我们将调查是否 膜回收率是相似的大和小的水平， 分泌，膜修复是否依赖于Ca 2+，以及是否 已知的激酶或磷酸酶在该过程中是重要的。因为我们是 作为为数不多的实时测量检索率的实验室之一， 我相信我们可以为理解这一点做出贡献， 重要的过程。 乙酰胆碱，从内脏神经末梢释放， 肾上腺髓质，通过激活Ca 2 +-去极化嗜铬细胞， 渗透性烟碱ACh受体，导致钙进入。ACh也 促进钙释放从细胞内储存，通过激活 毒蕈碱受体三种不同的Ca 2+信号相加触发 释放;这些是通过Ca 2+通道的钙进入，钙进入 通过Ca 2+渗透性烟碱ACh受体和钙释放 毒蕈碱乙酰胆碱受体激活后的细胞内储存。在 与绿色博士的合作，我们将研究相对重要性 以及它们如何协同工作。 N型钙通道几乎存在于所有神经元中。他们是 已知参与多种突触的神经递质释放 (as以及嗜铬细胞），但详细的生物物理信息 令人惊讶地难以获得，部分原因是N型Ca通道 也存在于其他类型的细胞中。我们有可用的克隆 N型钙通道在HEK细胞中稳定表达。协同 博士理查德米勒的实验室，我们计划进行广泛的研究， 通道门控电流。 这一拨款申请可能看起来很分散，但具体目标3和4是 加强我的实验室和 绿色博士和米勒博士的那些，所以应该在这个背景下进行判断。