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.

该应用重点关注胞吐作用和内吞作用的机制 嗜铬细胞。 Ca2+ 进入细胞后触发分泌 通过电压门控 Ca 通道。嗜铬细胞具有三种 不同类型的 Ca 通道。一种类型，促进钙通道， 不会被单次短暂的去极化激活。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 假设。 在我们迄今为止的所有实验中，儿茶酚胺释放之后是 快速膜检索（tau 秒）。我们将调查是否 对于大水平和小水平的膜回收率相似 分泌，膜回收是否依赖 Ca2+，以及是否 已知的激酶或磷酸酶在此过程中很重要。正如我们一样 作为少数几个实时测量检索率的实验室之一，我们 相信我们可以为理解这一点做出贡献 重要过程。 乙酰胆碱，从内脏神经末梢释放 肾上腺髓质，通过激活 Ca2+- 使嗜铬细胞去极化 可渗透的烟碱乙酰胆碱受体，导致钙进入。乙酰胆碱也 通过激活细胞内储存促进钙释放 毒蕈碱受体。三种不同的 Ca2+ 信号叠加以触发 发布;这些是通过 Ca2+ 通道的钙进入，钙进入 通过 Ca2+ 渗透性烟碱 ACh 受体和钙释放 毒蕈碱 ACh 受体激活后的细胞内储存。在 与格林博士合作，我们将调查相对重要性 每个信号的详细信息，以及它们如何协同工作。 几乎每个神经元中都发现了 N 型 Ca 通道。他们是 已知参与多种突触的神经递质释放 （以及嗜铬细胞），但详细的生物物理信息已经 令人惊讶的是很难获得，部分原因是 N 型 Ca 通道 存在于具有其他类型通道的细胞中。我们有可用的克隆 N型Ca通道在HEK细胞中稳定表达。与合作 理查德·米勒博士的实验室我们计划对 N 进行广泛的研究 通道选通电流。 该拨款申请可能看起来比较分散，但具体目标 3 和 4 是 加强我的实验室和 那些博士。格林和米勒等人都应该在这个背景下进行评判。