ION CHANNELS, CALCIUM AND NITRIC OXIDE IN VSM

描述(来自申请表的逐字记录)：这项建议旨在 找出血管SMC中的哪些不同通道负责 电容性(商店操作的)钙离子内流，以及它们是如何受到 商店。最近，我们描述了SMC和人类的电容钙内流(CCE) 并表明一氧化氮(NO)通过SERCA依赖抑制它 补给存储，但负责的离子通道的性质 对于SMC中的CCE，以及将通道激活和 对商店耗尽和补充的抑制仍然是完全的 未知。我们的初步数据显示，在细胞内耗尽时 在SMC中激活新的小(3ps)非选择性阳离子通道 取自兔和小鼠的大动脉。我们还发现，相同的频道可能是 由假定的钙内流因子(CIF)激活，该因子已部分地 从酵母和血小板中提纯出来的。我的建议的总体目标是 表征这些新的非选择性阳离子通道，建立它们的 SMC中容积性钙内流的功能作用及分子定位 它们的储存依赖的激活和抑制机制。主 这一假说认为，在血管SMC中，电容性钙离子内流是 由新的非选择性阳离子通道介导，该通道直接由一种 内质网分泌的钙内流因子(CIF) Ca~(2+)的耗竭储存并通过 胞吐作用样过程。抑制商店操作的钙内流可能会导致 因CIF生产、交付和/或CIF敏感度 商店经营的频道。这一假设将在Single的水平上进行检验 存储操作通道、全细胞电流、阳离子内流、细胞内钙离子 SMC和血小板产生的CIF。 本方案的具体目的是：目的1.确定SMC中的离子通道 负责商店操作的钙离子内流。我们将描述小说的特征 小的(3pS)非选择性阳离子通道，并通过 钙离子储存库的充盈状态。目的2.明确心绞痛的分子机制 SMC中的门店操作的通道调节。我们将确定是否 商店运营的通道直接由假设的CIF激活，该CIF由 SMC和血小板的内质网，还有什么已知物质可以 激活此通道，如果特定基质或与 细胞骨架是激活通道所必需的，如果磷酸酶抑制剂或 如果需要交付CIF，GTPGammaS可能会影响通道对CIF的敏感性 通过类似胞吐的过程激活细胞膜 需要锚定蛋白质。我们还将定义生理机制 门店渠道和CCE对CIF水平的抑制作用 单一渠道的生产、交付和CIF敏感性。

DESCRIPTION (Verbatim from the application): This proposal is aimed towards finding what distinction channels in vascular SMC are responsible for capacitative (store-operated) Ca2+ influx, and how they are regulated by the stores. Recently, we described capacitative Ca2+ entry (CCE) in SMC and human platelets and showed that nitric oxide (NO) inhibits it through SERCA-dependent refilling of the stores, but the nature of ion channels that are responsible for CCE in SMC, and the distinct mechanisms which link channel activation and inhibition to the depletion and refilling of the stores remains totally unknown. Our preliminary data showed that upon depletion of intracellular stores novel small (3 pS) nonselective cation channels are activated in SMC from rabbit and mouse aorta. We also found that the same channels could be activated by a putative calcium influx factor (CIF) that has been partially purified from yeast and platelets. The overall goal of my proposal is to characterize these novel nonselective cation channels, establish their functional role in capacitative Ca2+ influx in SMC, and to define the molecular mechanism of their store-dependent activation and inhibition. The main hypothesis of this proposal is that in vascular SMC capacitative Ca2+ entry is mediated by novel nonselective cation channels that are directly activated by a Ca2+ influx factor (CIF) that is produced by endoplasmic reticulum during depletion of Ca2+ stores and is delivered to the plasma membrane via exocytosis-like process. Inhibition of store-operated Ca2+ influx could result from impairment of CIF production, delivery, and/or CIF sensitivity of store-operated channels. This hypothesis will be tested at the level of single store-operated channels, whole-cell currents, cation influx, intracellular Ca2+ and CIF produced by SMC and platelets. Specific aims of this proposal are: Aim 1. To determine ion channels in SMC that are responsible for store-operated Ca2+ influx. We will characterize novel small (3 pS) nonselective cation channels and establish their regulation by the filling state of Ca2+ stores. Aim 2. To define the molecular mechanisms of store-operated channel regulation in SMC. We will determine if the store-operated channels are activated directly by a putative CIF produced by endoplasmic reticulum of SMC and platelets, what other known substances could activate this channel, if specific substrates or functional connection with cytoskeleton is required for channel activation, if phosphatase inhibitors or GTPgammaS could affect channel sensitivity to CIF, if CIF needs to be delivered to plasma membrane to activate the channels via exocytosis-like process requiring anchoring proteins. We will also define the physiological mechanisms of inhibition of store-operated channels and CCE on the level of CIF production, delivery and CIF-sensitivity of single channels.