Functional architecture of intracellular Ca2+ signals

描述(申请人提供)：激活细胞内钙离子通道引起的钙离子增加调节许多生理事件。这些钙通道的功能障碍涉及神经退行性疾病和神经系统疾病，以及外分泌、心脏异常和癌症。为了理解细胞功能是如何被钙信号控制的，以及病理信号是如何颠覆细胞功能的，我们必须了解细胞内钙通道的分布和属性(功能结构)是如何控制钙信号的模式的。在这里，我们研究了两个细胞内钙离子通透通道之间的结构和功能耦合，这两个通道由不同的第二信使激活：(I)烟酸腺嘌呤二核苷酸磷酸(NAADP)，激活最近发现的内溶酶体内的两孔通道(TPC)；(Ii)三磷酸肌醇(IP3)，激活内质网中的IP3受体(IP3Rs)。尽管定位于不同的细胞器，但这些钙通道的活性是密切相关的：在涉及溶酶体增殖的神经退行性疾病中，功能偶联受到越来越多的关注。在这里，通过定义哺乳动物的TPC相互作用组，我们提供了两个关键的未知因素：(I)TPC和IP3Rs之间的功能结构是如何建立的；(Ii)NAADP受体(NAADP-R，TPC复合体的一部分)的分子同一性。这两个都是设计新药以改变这种耦合的关键知识。我们的六人团队，结合了化学、蛋白质组、分子和活细胞成像的专业知识，将解决：(1)TPC是否是RAB效应器？TPC与一类Rab GTP酶相关。我们将定义TPC通道如何作为一个节点将钙离子信号耦合到内溶体运输和融合事件中。(2)TPC/IP3R活性在不同细胞器之间如何协调。我们将使用来自TPC相互作用组的新的分子洞察力来询问TPC/IP3Rs在膜接触部位的功能结构，并揭示调控失调如何触发溶酶体增殖。(3)NAADP-R的鉴定。我们设计并优化了一种新型的双功能光电探针来揭开TPC相互作用组中的NAADP-R。这是一个关键的障碍，阻碍了对TPC激活的了解。这项工作的广泛意义在于理解控制离子通道动力学的原理，从而理解控制细胞和系统水平划分结果的钙信号的动力学。这些数据将有助于我们理解无处不在的钙信号通路在健康和疾病中的作用。

DESCRIPTION (provided by applicant): Increases in Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological events. Dysfunction of these Ca2+ channels is involved in neurodegenerative and neurological disease, as well as exocrine, cardiac abnormalities and cancer. To appreciate how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must understand how both the distribution and the properties (the 'functional architecture') of intracellular Ca2+ channels controls the patterning of Ca2+ signals. Here, we investigate the structural and functional coupling between two intracellular Ca2+-permeable channels that are activated by distinct second messengers: (i) nicotinic acid adenine dinucleotide phosphate (NAADP) which activates the recently discovered two-pore channels (TPCs) within endolysosomes, and (ii) inositol trisphosphate (IP3), which activates IP3 receptors (IP3Rs) in the endoplasmic reticulum. Despite localization in separate organelles, the activity of these Ca2+ channels is intimately related: a functional coupling garnering increasing attention in neurodegenerative disorders involving lysosomal proliferation. Here, by defining the mammalian TPC interactome we provide insight into two key unknowns: (i) how the functional architecture between TPCs and IP3Rs is established and (ii) the molecular identity of the NAADP receptor (NAADP-R, part of the TPC complex). Both are key pieces of knowledge for designing new drugs to modify this coupling. Our six person team, combining chemical, proteomic, molecular and live cell imaging expertise, will resolve: (1) Whether TPCs are Rab effectors? TPCs associate with a clade of Rab GTPases. We will define how TPC channels act as a node for coupling Ca2+ signaling to endolysosomal trafficking and fusion events. (2) How TPC/IP3R activity is coordinated between discrete organelles. We will use novel molecular insight from the TPC interactome to interrogate the functional architecture of TPCs/IP3Rs at membrane contact sites, and uncover how dysregulation triggers lysosomal proliferation. (3) Identify the NAADP-R. We have designed and optimized a novel bifunctional photoprobe to unmask the NAADP-R within the TPC interactome. This is a key roadblock, hampering knowledge of TPC activation. The broad significance of this work is in understanding principles controlling ion channel dynamics, and thereby the kinetics of Ca2+ signals that control compartmentalized cellular and system-levels outcomes. Such data will aid our understanding of the role of ubiquitous Ca2+ signaling pathways in health and disease.