Wahl-Schott Christian, Freichel Marc, Hennis Konstantin, Philippaert Koenraad, Ottenheijm Roger, Tsvilovskyy Volodymyr, Varbanov Hristo
Institut für Kardiovaskuläre Physiologie und Pathophysiologie, Lehrstuhl für Vegetative Physiologie, Biomedizinisches Zentrum, Ludwig-Maximilians-Universität München, München, Germany.
Institute of Pharmacology, Heidelberg University, Heidelberg, Germany.
Handb Exp Pharmacol. 2023;278:277-304. doi: 10.1007/164_2023_637.
Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca measurements, (2) peri-endo-lysosomal Ca imaging using genetically encoded Ca sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca imaging by directing Ca indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca imaging.
内溶酶体是膜结合的酸性细胞器，参与细胞外和细胞内物质的内吞作用、再循环和降解。内溶酶体膜表达几种钙通透阳离子通道，包括双孔通道（TPC1 - 3）和瞬时受体电位黏脂蛋白通道（TRPML1 - 3）。在本章中，我们将描述四种不同的先进钙成像方法，这些方法非常适合研究内溶酶体阳离子通道的功能。这些技术包括：（1）全局胞质钙测量；（2）使用定向到胞质内溶酶体膜表面的基因编码钙传感器进行内溶酶体周围钙成像；（3）对经过工程改造以便与方法1和2结合将其重定向到质膜的内溶酶体阳离子通道进行钙成像；（4）通过将钙指示剂定向到内溶酶体腔进行钙成像。此外，我们将综述有用的小分子，它们可作为内溶酶体钙成像的宝贵工具。我们将讨论与内溶酶体钙成像相关的特定方法学问题，而不是提供完整的方案。
