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

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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.