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; 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.

该应用的重点是胞吐作用和内吞作用的机制 铬蛋白细胞。分泌是由Ca2+进入细胞的触发的 通过电压门控CA通道。铬蛋白细胞拥有三个 不同类型的CA通道。一种类型，促进CA渠道， 不会被单个短暂去极化激活。招募这些 通道可能涉及一种新型的通道调节形式，电压 - 依赖性磷酸化。正在进行的研究将验证（或反驳） 假设。 在迄今为止的所有实验中，儿茶酚胺的释放之后是 快速膜检索（秒的tau）。我们将调查是否 膜的检索率相似 分泌，膜检索是否为Ca2+依赖性，以及是否 已知的激酶或磷酸酶在此过程中很重要。就像我们一样 实时测量检索率的少数实验室之一，我们 相信我们为理解这一点做出了贡献 重要过程。 乙酰胆碱，从诊断神经终端释放 肾上腺髓质，通过激活Ca2+ - 去极化铬蛋白细胞去极化 可渗透的烟碱ACH受体，导致钙进入。 ACH也 通过激活从细胞内存储中促进钙释放 毒蕈碱受体。三个不同的Ca2+信号总结到触发 发布;这些是通过Ca2+通道进入钙的钙进入钙 - 进入 通过Ca2+ - 可渗透的烟碱ACH受体和钙释放 毒蕈碱ACH受体激活后的细胞内存储。在 与格林博士的合作，我们将研究相对重要性 这些信号中的每一个以及它们如何一起工作。 几乎在每个神经元中都发现了N型CA通道。他们是 已知在各种突触中参与神经递质释放 （以及染色体细胞），但详细的生物物理信息具有 很难获得，部分原因是N型CA通道 在具有其他类型的通道的细胞中发现。我们有克隆 N型Ca通道在HEK细胞中稳定表达。与 理查德·米勒（Richard Miller）博士实验室，我们计划对N进行广泛的研究 通道门控电流。 该赠款应用可能看起来分散，但特定的目标3和4是 协作努力加强了我的实验室与 那些博士。绿色和米勒等应在这种情况下进行判断。