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)容量性钙内流的离子通道，并建立其储存依赖的调节机制。根据我们最近的研究，为我们提供了新的令人兴奋的可能性来确定SMC中天然商店操作的阳离子通道的分子身份，但它们需要额外的资金。最近，在确定了钙离子内流在激动剂诱导的SMC收缩和一氧化氮诱导的SMC松弛中的生理重要性后，我们成功地描述了一种新的小电导(3ps)非选择性阳离子通道，该通道在钙离子储备耗尽时被激活，并与SMC的容积性钙内流有关。我们还证实了它是由钙内流因子(C1F)直接激活的。尽管这种储存操作的阳离子通道(SOC)非常重要，但它的分子同一性尚不清楚。Trp通道家族中的一些成员(或它们的组合)被认为参与了钙离子内流，但这些数据存在很大争议，目前还不清楚它们是否与SMC中的SOC通道有关。我们的初步研究强烈表明SOC和Trp通道之间存在功能联系。事实上，我们发现钙调蛋白(CaM)抑制我们的天然SOC通道，类似于最近对Trp通道的描述。我们还发现，已知的7个TRPC通道中有3个存在于主动脉SMC中(即TRPC1、TRPC3和TRPC6)。伯恩鲍默博士刚刚完成了两只缺乏TRPC1或TRPC6基因的敲除小鼠的培育(TRPC3-/-小鼠正在进行中)，我们可以培育出每种类型的繁殖夫妇。利用这些转基因模型，我们有一个独特的机会来确定TRPC1、TRPC6和/或TRPC3基因产物是否可能是血管SMC天然SOC的一部分。本补充方案的具体目的是验证TRPCI、TRPC6和/或TRPC3基因产物可能是血管SMC天然存储操作通道的功能部分的假设。这一假说将用电生理学、成像、分子和生化方法在单个存储操作通道、全细胞电流、细胞内钙离子和血管SMC蛋白表达水平以及对照和转基因TRPI-/-、TRP6-I-和TRP3-I-MICE的血管收缩和松弛水平上进行验证。原生SOC通道功能中的TRPC6和TRPC3。我们将首先确认TRPC1-/-和TRPC6-/-小鼠的主动脉SMC中TRPCI或TRPC6基因的靶向性中断，并确定在TRPC1-/-和TRPC6-/-小鼠的SMC中，CJF和CaM依赖的3ps通道调节、容量钙内流和收缩-松弛的性质和机制是否有任何差异。同样的研究将在TRPC3-/-小鼠的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)