Mallmann Robert T, Gonzalez Mantuano Marlene C, Polomski Katharina, Knerr Julian, Klugbauer Norbert
Institute for Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
Institute for Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
J Biol Chem. 2025 Sep;301(9):110576. doi: 10.1016/j.jbc.2025.110576. Epub 2025 Aug 8.
Two-pore channels (TPCs) constitute a small family of cation channels expressed in endo-lysosomal compartments. TPCs have been characterized as important constituents controlling Ca-mediated vesicular membrane fusion and fission, thereby regulating intracellular organelle trafficking. Two activators, nicotinic acid adenine dinucleotide phosphate and phosphatidylinositol-3,5-bisphosphate, induce ion flux through TPCs. The membrane-permeable small molecule activators TPC2-A1-N and TPC2-A1-P have been identified and postulated to mimic their action and to discriminate for a preferential selectivity either for Ca or for Na. This was observed only for TPC2 and was independent of nicotinic acid adenine dinucleotide phosphate-binding proteins. Here, we applied TPC2-A1-N and measured intracellular increase of Ca and Na in mouse embryonic fibroblast, HeLa, and J774 cells. TPC2-A1-N did not only increase Ca levels in WT but also in all cells with genetically inactivated TPCs. Depletion of Ca from the endoplasmic reticulum (ER) via thapsigargin caused a massive reduction of the TPC2-A1-N induced Ca elevation in all cell lines, indicating that ER plays a key role in this context. Furthermore, our results point to an inositol triphosphate receptor-independent TPC2-A1-N mediated Ca release. Ca depletion from ER was also observed by using an ER-targeted GCaMP6 construct. TPC2-A1-N also raised Na levels in mouse embryonic fibroblast cells deficient for TPC1 and TPC2. In summary, our results suggest that TPC2-A1-N induced Ca and Na signals are independent of any TPC and that ER represents the major source of Ca.
双孔通道（TPCs）构成了一个在内涵体 - 溶酶体区室中表达的阳离子通道小家族。TPCs已被确定为控制钙介导的囊泡膜融合与裂变的重要成分，从而调节细胞内细胞器运输。两种激活剂，烟酰胺腺嘌呤二核苷酸磷酸（NAADP）和磷脂酰肌醇 - 3,5 - 二磷酸（PI(3,5)P2），可诱导离子通过TPCs流动。已鉴定出膜通透性小分子激活剂TPC2 - A1 - N和TPC2 - A1 - P，并推测它们模拟了NAADP和PI(3,5)P2的作用，且对钙或钠具有优先选择性。这仅在TPC2中观察到，且与NAADP结合蛋白无关。在此，我们应用TPC2 - A1 - N并测量了小鼠胚胎成纤维细胞、HeLa细胞和J774细胞内钙和钠的增加情况。TPC2 - A1 - N不仅增加了野生型细胞中的钙水平，还增加了所有TPC基因失活细胞中的钙水平。通过毒胡萝卜素从内质网（ER）中耗尽钙，导致所有细胞系中TPC2 - A1 - N诱导的钙升高大幅降低，表明在内质网在这种情况下起关键作用。此外，我们的结果表明存在一种不依赖于肌醇三磷酸受体的TPC2 - A1 - N介导的钙释放。使用内质网靶向的GCaMP6构建体也观察到了内质网钙的耗尽。TPC2 - A1 - N还提高了缺乏TPC1和TPC2的小鼠胚胎成纤维细胞中的钠水平。总之，我们的结果表明TPC2 - A1 - N诱导的钙和钠信号不依赖于任何TPC，且内质网是钙的主要来源。
