ION CHANNELS, CALCIUM AND NITRIC OXIDE IN VSM

描述（来自申请的逐字记录）：本提案旨在 发现血管SMC中的哪些不同通道负责 容量性（存储操作）Ca 2+内流，以及它们如何被调节， 店最近，我们描述了在SMC和人类的容量性Ca 2+内流（CCE）， 血小板，并表明一氧化氮（NO）抑制它通过SERCA依赖性 但负责离子通道的性质 对于SMC中的CCE，以及将通道激活和 对贮存物的消耗和补充的抑制仍然完全存在 未知我们的初步数据表明，当细胞内 SMC中的新型小（3 pS）非选择性阳离子通道被激活 从兔子和老鼠的主动脉。我们还发现，同样的渠道， 被一种假定的钙内流因子（CIF）激活， 从酵母和血小板中提纯。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 在SMC中的容量性Ca 2+内流中的功能作用，并确定分子 其存储依赖性激活和抑制的机制。主要 这一建议的假设是，在血管SMC中， 由新的非选择性阳离子通道介导， Ca 2+内流因子（CIF），由内质网在 消耗Ca 2+储存，并通过 胞吐样过程。抑制钙池操作的Ca 2+内流可能导致 CIF生产、交付和/或CIF敏感性的减值 商店运营的渠道。这一假设将在单一的水平上进行检验。 储存操纵通道、全细胞电流、阳离子内流、细胞内Ca 2 + 由SMC和血小板产生CIF。 本提案的具体目标是：目标1。测定SMC中的离子通道 负责钙池操作的钙离子内流。我们将描述小说 小的（3 pS）非选择性阳离子通道，并通过 钙库的充盈状态。目标二。来确定 SMC中的商店运营渠道管理。我们将确定 存储操作的通道直接由假定的CIF激活，CIF由 SMC和血小板的内质网，还有什么已知的物质可以 激活这个通道，如果特定的底物或功能连接， 细胞骨架是通道激活所必需的，如果磷酸酶抑制剂或 如果需要输送CIF，GTP γ S可能会影响通道对CIF的敏感性 通过胞吐样过程激活通道 需要锚定蛋白。我们还将定义 在CIF水平上抑制商店运营渠道和CCE 单渠道的生产、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.