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

TPCs（双孔通道）构成了一个小的阳离子通道家族，在内溶酶体系统的膜中表达。TPCs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TPCs结合，而是与通道复合体的其他蛋白质结合。最近，在文献中提出了两种候选蛋白作为naadp结合成分，它们激活各种模型系统中的tpc并诱导Ca2+电流。在此应用范围内，我们希望实现三个目标：(1)通过使用CRISPR/ cas方法，我们将建立基于细胞的naadp结合蛋白遗传模型。我们将进行细胞内Ca2+测量，以研究TPCs在其天然膜室中naadp活化的机制。NAADP将通过应用新建立的技术包装到脂质体中，并添加到MEF细胞（WT, TPC1-， TPC2-和TPC1/2- double - ko）中进行Fura2 Ca2+测量。我们将应用naadp结合和TPC蛋白的操作，如敲除、突变或过表达成分，并将研究它们的细胞（co）定位。(2)在之前的资助期间，我们进行了详细的RNAseq分析，从而对TPC缺陷细胞的转录组改变有了新的认识。多种膜受体表达不同，其信号通路受到影响。现在，我们打算关注选定受体的内吞作用和细胞内运输过程的机制。我们将研究TPC缺陷细胞中小泡蛋白表达强烈降低对内吞作用、受体运输和受体信号通路的影响。标记的rab - gtpase将用于内溶酶体囊泡的显微活细胞成像。(3)基于我们对TPCs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。我们最近开始了抗原诱导关节炎（AIA）的模型，该模型将进一步发展。此外，我们将建立慢性炎症性肠病模型，右旋糖酐硫酸钠（DSS）诱导结肠炎。

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.