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

描述(由申请人提供)：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钙信号第二信使，已被报道从酸性钙库或溶酶体中动员钙离子来触发全球钙反应。然而，目前尚不清楚该信号核苷酸如何激活溶酶体的钙释放，以及该细胞器上NAADP敏感的钙释放通道的特性。目前的提议将验证一个中心假设，即冠状动脉平滑肌细胞(CASMCs)溶酶体中对NAADP敏感的钙释放通道具有瞬时受体电位-粘蛋白1(Trp-ML1)的特征，它介导溶酶体局部的钙爆发，并导致参与冠状动脉对激动剂的血管收缩反应的两相钙释放。为了验证这一假设，本文提出了四个具体目标。具体目的1利用脂类双层通道重构技术对CASMC的溶酶体钙释放通道进行表征，并探讨其与钙或氢敏感性相关的调控机制。特定目的2试图通过不同的方法，包括生化和分子检测、电生理方法、共聚焦显微镜、FRET和Trp-ML1缺陷细胞的使用，来证明在纯化的溶酶体和完整的冠状动脉心肌细胞中，NAADP敏感的钙释放通道是Trp-ML1。在具体目标3中，我们将用电子显微镜鉴定CASMCs中溶酶体和SR之间的结构和功能连接，并用全内反射显微镜探索两个细胞器通过这个连接相互作用的机制。最后，通过对加压小冠状动脉的视频显微镜观察，明确Trp-ML1作为NAADP敏感的钙释放通道是否有助于调节血管张力和血管运动反应。这些研究将首次将NAADP诱导的溶酶体钙释放与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钙水平中的新功能。这些发现将显著增加我们对细胞内钙信号的重要时空调节与溶酶体钙存储或两池机制公共卫生相关性的理解：这项拨款提案试图研究一种新的信号机制，通过这种机制来调节心脏动脉壁肌肉细胞内的钙浓度。虽然有一些报告表明，这些细胞内的钙通过两个阶段的释放过程释放出来，导致动脉收缩，但这种两个阶段的释放是如何发生的还不清楚。目前的拨款提案将证明一种被称为瞬时受体潜力的蛋白质-粘蛋白1作为触发器，在细胞内介导这种两相钙释放。这一发现将有助于我们理解导致心脏动脉收缩的机制，因此可能有助于开发新的冠心病治疗方法，以防止未来的心脏病发作。

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.