Ion Channels, calcium regulation and nitric oxide in vsm

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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)