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）作为一种新的和有效的细胞内Ca2+信号第二信使，可以从酸性Ca2+储存或溶酶体中动员Ca2+，从而触发全局Ca2+反应。然而，目前尚不清楚这种信号核苷酸如何激活溶酶体中的Ca2+释放，以及该细胞器上naadp敏感的Ca2+释放通道的身份。本提案将验证一个中心假设，即冠状动脉平滑肌细胞（CASMCs）溶酶体中naadp敏感的Ca2+释放通道是瞬时受体电位-黏脂素1 （TRP-ML1）的特征，该通道介导溶酶体的局部Ca2+爆发，并导致两期Ca2+释放参与冠状动脉对激动剂的血管运动反应。为了验证这一假设，提出了四个具体目标。特异性Aim 1将通过脂质双分子层通道重构技术表征CASMCs溶酶体Ca2+释放通道，然后探索调节这些通道与Ca2+或H+敏感性相关的机制。特异性目的2试图证明naadp敏感的Ca2+释放通道是TRP-ML1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-ML1缺陷细胞。在Specific Aim 3中，我们将使用电子显微镜识别CASMCs中溶酶体和SR之间的结构和功能连接，并使用全内反射显微镜探索两个细胞器通过这种连接相互作用的机制。最后，Specific Aim 4将通过分离的加压小冠状动脉视频显微镜确定TRP-ML1作为naadp敏感的Ca2+释放通道是否有助于血管张力和血管舒张反应的调节。这些拟议的研究将首次将naadp诱导的溶酶体Ca2+释放与TRP-ML1通道联系起来，从而揭示溶酶体TRP-ML1在调节血管平滑肌细胞内Ca2+水平中的新功能。这些发现将显著增加我们对与溶酶体Ca2+储存或双池机制相关的细胞内Ca2+信号的重要时空调节的理解。公共卫生相关性：本拨款提案旨在研究一种新的信号机制，通过该机制调节心脏动脉壁肌肉细胞内的钙浓度。尽管有一些报道表明这些细胞内的钙通过两阶段释放过程被释放以产生动脉收缩，但尚不清楚这两阶段释放是如何发生的。目前的拨款提案将证明一种被称为瞬时受体电位粘脂蛋白1的蛋白质作为触发因子介导细胞内钙的两相释放。这些发现将有助于我们理解心脏动脉收缩的机制，因此可能有助于开发冠状动脉疾病的新疗法，以预防未来的心脏病发作。

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 PUBLIC HEALTH RELEVANCE: This grant proposal seeks to study a novel signaling mechanism by which calcium concentrations within muscle cells of the heart artery wall are regulated. Although there are some reports indicating that calcium within these cells are released to produce contraction of the arteries through a two-phase release process, it is unknown how this two-phase release happens. The present grant proposal will demonstrate that a protein called as transient receptor potential-mucolipin1 serves as a trigger to mediate this two- phase calcium release within the cells. The findings will importantly contribute to our understanding of the mechanisms responsible for contraction of the heart arteries and therefore may help develop new therapy for coronary arterial disease to prevent heart attack in the future.