Ion Channels, calcium regulation and nitric oxide in vsm

描述（由申请人提供）：我的长期目标是确定负责血管平滑肌细胞（SMC）中容量性Ca 2+内流的离子通道，并建立其储存依赖性调节机制。根据我们最近的研究，新的令人兴奋的可能性为我们打开，以确定SMC中的本地存储操作的阳离子通道的分子身份，但他们需要额外的资金。最近，在建立了平滑肌细胞中激动剂诱导的收缩和一氧化氮诱导的舒张中钙池操纵的Ca 2+内流的生理重要性之后，我们成功地表征了一种新的小电导（3 pS）非选择性阳离子通道，它在钙库耗尽时被激活，并负责SMC中的容量性钙内流。我们还确定了它直接被钙内流因子（C1 F）激活。尽管这种存储操作的阳离子（SOC）通道的巨大的重要性，其分子身份是未知的。TRP通道家族的一些成员（或其组合）与钙池操作的Ca 2+内流有关，但这些数据存在很大争议，目前尚不清楚它们是否与SMC中的SOC通道相关。我们的初步研究强烈建议SOC和TRP通道之间的功能联系。事实上，我们发现钙调素（CaM）抑制我们的天然SOC通道，类似于最近描述的TRP通道。我们还发现，七个已知的TRPC通道中有三个存在于主动脉SMC中（即TRPC 1，TRPC 3和TRPC 6）。Birnbaumer博士刚刚完成了两个缺乏TRPC 1或TRPC 6基因的敲除小鼠的开发（TRPC 3-/-小鼠正在路上），每种类型的育种夫妇都可供我们使用。使用这些转基因模型，我们有一个独特的机会，以确定是否TRPC 1，TRPC 6和/或TRPC 3基因产物可能是一个天然SOC在血管SMC的一部分。本补充提案的具体目标是测试的假设，TRPC 1，TRPC 6和/或TRPC 3基因产物可能是一个功能部分的天然存储操作的通道在血管SMC。将使用电生理学、成像、分子和生物化学方法在单个储存操纵的通道、全细胞电流、血管SMC中的细胞内Ca 2+和蛋白质表达以及对照和转基因TRPI-/-、TRP 6-I-和TRP 3-I-小鼠中的血管收缩和舒张的水平上测试该假设。TRPC 6和TRPC 3在原生SOC通道功能中。我们将首先确认TRPC 1-/-和TRPC 6-/-小鼠主动脉SMC中TRPC 1或TRPC 6基因的靶向破坏，并确定是否存在CJF和CaM依赖性调控储存操纵的3 pS通道的性质和机制的任何差异，TRPC-/-和TRPC 6-/-小鼠SMC的容性Ca ~（2+）内流和收缩-舒张功能，TRPC 3-/-小鼠SMC的容性Ca ~（2+）内流和收缩-舒张功能也将在TRPC 3-/-小鼠SMC中进行（预计在未来6-12个月内）

DESCRIPTION (provided by the applicant): My long-term goal is to define ion channels that are responsible for capacitative Ca2+ influx in vascular smooth muscle cells (SMC) and to establish the mechanism of their store-dependent regulation. Based on our recent studies, new exciting possibilities opened for us to determine the molecular identity of native store-operated cation channels in SMC, but they require additional funds. This is a reason for proposing this study as a supplement to my existing R0l grant.Recently, after establishing the physiological importance of store-operated Ca2+ influx in agonist-induced contraction and nitric oxide-induced relaxation in SMC, we successfully characterized a novel small conductance (3 pS) nonselective cation channel, that is activated upon the depletion of Ca2+ stores and is responsible for capacitative Ca2+ influx in SMC. We also established that it is activated directly by calcium influx factor (C1F). In spite of the tremendous importance of this store-operated cation (SOC) channel, its molecular identity is unknown. Some of the members of the family of TRP channels (or their combination) have been implicated in store-operated Ca2+ entry, but the data are highly controversial, and it is still unclear if they are relevant to SOC channels in SMC. Our preliminary studies strongly suggest a functional link between SOC and TRP channels. Indeed, we found that calmodulin (CaM) inhibits our native SOC channel, similar to what was described recently for TRP channels. We also found that three out of seven known TRPC channels are present in aortic SMC (namely TRPC1, TRPC3 and TRPC6). Dr. Birnbaumer just completed the development of two knock-out mice lacking TRPC1 or TRPC6 genes (TRPC3-/- mice are on the way), and breeding couples of each kind are available for us. Using these transgenic models we have a unique opportunity to determine if TRPC1, TRPC6 and/or TRPC3 gene products could be a part of the native SOC in vascular SMC.The specific goal of this supplemental proposal is to test the hypothesis that TRPCI, TRPC6 and/or TRPC3 gene products could be a functional part of native store-operated channel in vascular SMC. This hypothesis will be tested using electrophysiological, imaging, molecular and biochemical approaches on the level of single store-operated channels, whole-cell currents, intracellular Ca2+ and protein expression in vascular SMC, as well as vascular contraction and relaxation in control and transgenic TRPI-/-, TRP6-I- and TRP3-I- mice.Specific Aim of this supplemental proposal is:AIM 1: To determine the role of TRPC1. TRPC6 and TRPC3 in native SOC channel function. We will first confirm the targeted disruption of TRPCI or TRPC6 genes in aortic SMC from TRPC1-/- and TRPC6-/- mice, and determine if there are any differences in the properties and mechanism of CJF- and CaM-dependent regulation of store-operated 3 pS channels, capacitative Ca2+ influx and contraction- relaxation in SMC from TRPCI-/- and TRPC6-/- mice.The same studies will be done in SMC of TRPC3-/- mice when it becomes available (expected within the next 6-12 months)