NAADP-Sensitive Lysosomal Ca2+ Release Channels-TRP-ML1 in Arterial Myocytes

描述（由申请人提供）：据报道，烟酸腺嘌呤二核苷酸磷酸（NAADP）作为一种新型有效的细胞内Ca 2+信号传导第二信使，可从酸性Ca 2+库或溶酶体中动员Ca 2+，以触发整体Ca 2+反应。然而，这仍然是未知的信号核苷酸如何激活从溶酶体的Ca 2+释放和什么是NAADP敏感的Ca 2+释放通道上这个细胞器的身份。目前的建议将测试一个中心的假设，即冠状动脉平滑肌细胞（CASMCs）的溶酶体中的NAADP敏感的Ca 2+释放通道是瞬时受体电位-粘磷脂1（TRP-ML 1）的特征，它介导局部Ca 2+从溶酶体爆发，并导致两相Ca 2+释放参与冠状动脉对激动剂的血管反应。为了验证这一假设，提出了四个具体目标。具体目标1将通过脂质双层通道重建技术表征CASMCs的溶酶体Ca 2+释放通道，并探讨这些通道与Ca 2+或H+敏感性相关的调控机制。具体目标2试图证明身份NAADP敏感的钙释放通道是TRP-ML 1在纯化的溶酶体和完整的冠状动脉肌细胞使用不同的方法，包括生化和分子检测，电生理方法，共聚焦显微镜，FRET，和使用TRP-ML 1缺陷细胞。在具体目标3中，我们将使用电子显微镜确定CASMC中溶酶体和SR之间的结构和功能连接，并通过使用全内反射显微镜探索两个细胞器通过该连接相互作用的机制。最后，具体目标4将确定TRP-ML 1作为NAADP敏感的Ca 2+释放通道是否有助于通过视频显微镜观察孤立的加压小冠状动脉来调节血管张力和血管收缩反应。这些拟议的研究将首次将NAADP诱导的溶酶体Ca 2+释放与TRP-ML 1通道联系起来，从而揭示了溶酶体TRP-ML 1在调节血管平滑肌细胞胞内Ca 2+水平中的新功能。这一发现将大大增加我们对与溶酶体钙库或两池机制相关的细胞内钙信号的重要时空调节的理解

DESCRIPTION (provided by applicant): Nicotinic acid adenine dinucleotide phosphate (NAADP) as a novel and potent intracellular Ca2+ signaling second messenger has been reported to mobilize Ca2+ from an acidic Ca2+ store or lysosomes to trigger a global Ca2+ response. However, it remains unknown how this signaling nucleotide activates Ca2+ release from lysosomes and what is the identity of NAADP-sensitive Ca2+ release channels on this organelle. The present proposal will test a central hypothesis that an NAADP-sensitive Ca2+ release channel in lysosomes of coronary arterial smooth muscle cells (CASMCs) is characteristic of transient receptor potential-mucolipin1 (TRP-ML1), which mediates local Ca2+ bursts from lysosomes and leads to a two-phase Ca2+ release participating in the vasomotor response of coronary arteries to agonists. To test this hypothesis, four Specific Aims are proposed. Specific Aim 1 will characterize lysosomal Ca2+ release channels from CASMCs by lipid bilayer channel reconstitution technique and then explore the mechanisms regulating these channels related to Ca2+ or H+ sensitivity. Specific Aim 2 attempts to demonstrate the identity of NAADP-sensitive Ca2+ release channels to be TRP-ML1 in purified lysosomes and in intact coronary arterial myocytes using different approaches including biochemical and molecular detections, electrophysiological approaches, confocal microscopy, FRET, and use of TRP-ML1 deficient cells. In Specific Aim 3, we will identify the structural and functional junction between the lysosomes and SR in CASMCs using electron microscopy and to explore the mechanisms by which two organelles interact via this junction by using total internal reflectance microscopy. Finally, Specific Aim 4 will determine whether TRP-ML1 as a NAADP-sensitive Ca2+ release channel contributes to the regulation of vascular tone and vasomotor response by video microscopy of isolated pressurized small coronary arteries. These proposed studies will, for the first time, link NAADP-induced lysosomal Ca2+ release to TRP-ML1 channels and thereby reveal a novel function of lysosomal TRP- ML1 in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells. The findings will significantly increase our understanding of an important spatiotemporal regulation of intracellular Ca2+ signaling associated with the lysosomal Ca2+ store or two- pool mechanism