STUDIES OF EXOCYTOSIS AND ENDOCYTOSIS

这一应用主要集中在胞吐和胞吞的机制上。 嗜铬细胞。分泌是由钙离子进入细胞触发的 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， 不会被一次短暂的去极化激活。招募这些人 通道可能涉及一种新形式的通道调节，电压- 依赖的磷酸化。正在进行的研究将证实(或反驳)这一点 假设。 在我们迄今为止的所有实验中，儿茶酚胺的释放紧随其后 快速膜检索(T秒)。我们将调查是否 对于大水平和小水平的膜检索速度是相似的 分泌，膜恢复是否依赖于钙离子，以及 已知的激酶或磷酸酶在这一过程中很重要。就像我们一样 作为为数不多的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 重要的过程。 从内脏神经末梢释放的乙酰胆碱 肾上腺髓质，通过激活钙离子使嗜铬细胞去极化 通透性烟碱型ACh受体，导致钙内流。ACH也是 通过激活促进细胞内储存的钙释放 毒鼠碱受体。三种不同的钙信号相加触发 释放；这些是通过钙离子通道的钙内流，钙内流 通过钙离子通透性烟碱受体和钙释放 毒扁豆碱受体激活后的细胞内储存。在……里面 与格林博士合作，我们将调查 这些信号中的每一个以及它们如何协同工作。 几乎在每个神经元中都发现了N型钙通道。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 出人意料地难以获得，部分原因是N型钙通道 在具有其他类型通道的细胞中被发现。我们有可用的克隆 N-型钙通道在HEK细胞中稳定表达。与 理查德·米勒博士的实验室我们计划对N 通道门控电流。 这项拨款申请可能看起来很分散，但具体的目标3和4是 协作努力，加强我的实验室和 格林博士和米勒博士等人的观点应该在这一背景下加以评判。

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.