IP₃ Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca²⁺ to Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) allow extracellular stimuli to redistribute Ca²⁺ from the ER to cytosol or other organelles. We show, using small interfering RNA (siRNA) and vacuolar H⁺-ATPase (V-ATPase) inhibitors, that lysosomes sequester Ca²⁺ released by all IP₃R subtypes, but not Ca²⁺ entering cells through store-operated Ca²⁺ entry (SOCE). A low-affinity Ca²⁺ sensor targeted to lysosomal membranes reports large, local increases in cytosolic [Ca²⁺] during IP₃-evoked Ca²⁺ release, but not during SOCE. Most lysosomes associate with endoplasmic reticulum (ER) and dwell at regions populated by IP₃R clusters, but IP₃Rs do not assemble ER-lysosome contacts. Increasing lysosomal pH does not immediately prevent Ca²⁺ uptake, but it causes lysosomes to slowly redistribute and enlarge, reduces their association with IP₃Rs, and disrupts Ca²⁺ exchange with ER. In a "piston-like" fashion, ER concentrates cytosolic Ca²⁺ and delivers it, through large-conductance IP₃Rs, to a low-affinity lysosomal uptake system. The involvement of IP₃Rs allows extracellular stimuli to regulate Ca²⁺ exchange between the ER and lysosomes.