Characterization of Endo-Lysosomal Cation Channels Using Calcium Imaging

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 Ca²⁺-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 Ca²⁺ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca²⁺ measurements, (2) peri-endo-lysosomal Ca²⁺ imaging using genetically encoded Ca²⁺ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca²⁺ 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) Ca²⁺ imaging by directing Ca²⁺ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca²⁺ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca²⁺ imaging.