Two-pore channels (TPCs): NAADP-activation mechanisms and function for intracellular transport processes

TPC(两孔通道)构成了一个小的阳离子通道家族，表达于内溶酶系统的膜上。TPC通过第二信使烟酸腺嘌呤二核苷酸磷酸(NAADP)激活。然而，NAADP并不直接与TPC结合，而是与通道复合体的其他蛋白质结合。最近，文献中提出了两种候选蛋白作为NAADP结合组分，它们激活不同模型系统中的TPC，并诱导钙电流。在本申请的范围内，我们希望实现三个目标：(1)利用CRISPR/CAS方法，我们将建立NAADP结合蛋白的基于细胞的遗传模型。我们将进行细胞内钙离子的测量，以研究TPC在其自然膜室中NAADP激活的机制。NAADP将通过应用一项新建立的技术被包装成脂质体，并添加到MEF细胞(WT、TPC1-、TPC2-和TPC1/2-Double-KO)中用于Fura2钙测量。我们将应用NAADP结合和TPC蛋白的操作，如基因敲除、突变或过度表达的成分，并对它们的细胞(共)定位进行调查。(2)在前一个资助期间，我们进行了详细的RNAseq分析，使我们能够对TPC缺陷细胞的改变的转录组有新的见解。多种膜受体的表达存在差异，其信号转导途径受到影响。现在，我们打算重点研究选定受体的内吞作用和细胞内转运过程的机制。我们将研究TPC缺陷细胞中小窝蛋白表达显著减少对内吞作用、受体转运和受体信号通路的影响。标记的Rab-GTP酶将用于内溶酶体小泡的显微活细胞成像。(3)基于我们对TPC在巨噬细胞释放细胞因子中的作用的研究，我们将建立自身免疫性疾病的体内模型。我们最近开始了一个抗原诱导性关节炎(AIA)的模型，该模型将进一步发展。此外，我们还将建立一种慢性炎症性肠病模型，即葡聚糖硫酸钠(DSS)诱导的结肠炎。

TPCs (two-pore channels) constitute a small family of cation channels that are expressed in membranes of the endolysosomal system. TPCs are activated via the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). However, NAADP does not directly bind to TPCs, instead to other proteins of the channel complex. Recently, in literature two candidate proteins have been suggested as NAADP-binding components, which activate TPCs in various model systems and which induce a Ca2+ current. Within the scope of this application we want to achieve three objectives:(1) By using the CRISPR/Cas-method we will establish cell-based genetic models of the NAADP-binding proteins. We will perform intracellular Ca2+ measurements to investigate the mechanisms of NAADP-activation of TPCs in their natural membrane compartment. NAADP will be packed into liposomes by applying a newly established technique and added to MEF cells (WT, TPC1-, TPC2- and TPC1/2-Double-KO) for Fura2 Ca2+ measurements. We will apply manipulations of the NAADP-binding and the TPC proteins such as knockouts, mutants or overexpressed components and their cellular (co)-localization will be investigated.(2) During the previous funding period we performed detailed RNAseq analyses which allowed new insights into the altered transcriptome of TPC deficient cells. Numerous membrane receptors were differently expressed and their signaling pathways were affected. Now we intend to focus on the mechanisms of endocytosis and intracellular transport processes of selected receptors. We will investigate the consequences of the strongly reduced expression of caveolins in TPC deficient cells for endocytosis, for receptor trafficking and for receptor signaling pathways. Labelled Rab-GTPases will be used for microscopic live-cell-imaging of endolysosomal vesicles.(3) Based on our work for the role of TPCs for the release of cytokines from macrophage cell lines, we will establish in vivo models of autoimmune diseases. We recently started with a model for antigen-induced arthritis (AIA), which will be developed further. Additionally, we will establish a model for chronic inflammatory bowel diseases, the Dextran Sodium Sulfate (DSS) induced colitis.