Functional architecture of intracellular Ca2+ signals

描述（由申请人提供）：细胞内Ca2+通道激活引起的Ca2+增加调节许多生理事件。这些Ca2+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。为了了解细胞功能是如何被Ca2+信号控制的，以及病理线索是如何破坏它们的功能的，我们必须了解细胞内Ca2+通道的分布和特性（“功能结构”）是如何控制Ca2+信号的模式的。在这里，我们研究了由不同的第二信使激活的两个细胞内Ca2+可渗透通道之间的结构和功能偶联：(i)激活内溶酶体内最近发现的双孔通道（TPCs）的烟酸腺嘌呤二核苷酸磷酸（NAADP）和（ii）激活内质网IP3受体（IP3Rs）的肌醇三磷酸（IP3）。尽管在不同的细胞器中定位，这些Ca2+通道的活性密切相关：在涉及溶酶体增殖的神经退行性疾病中，功能偶联越来越受到关注。在这里，通过定义哺乳动物TPC相互作用组，我们提供了对两个关键未知的见解：(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）的分子身份。两者都是设计改变这种耦合的新药的关键知识。我们的六人团队，结合化学，蛋白质组学，分子和活细胞成像专业知识，将解决：(1)TPCs是否是Rab效应物？TPCs与拉布gtp酶的一个分支有关。我们将定义TPC通道如何作为Ca2+信号耦合到内溶酶体运输和融合事件的节点。(2) TPC/IP3R活性如何在离散细胞器之间协调。我们将使用来自TPC相互作用组的新分子见解来询问膜接触部位的TPC /IP3Rs的功能结构，并揭示失调如何触发溶酶体增殖。(3)识别NAADP-R。我们设计并优化了一种新型双功能光探针来揭示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，从而控制区隔细胞和系统水平结果的Ca2+信号动力学。这些数据将有助于我们理解无处不在的Ca2+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.