SummaryInositol 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.

The dipeptide glycyl- l -phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pH ly ), and transient increases in cytosolic pH (pH cyt ) and Ca 2+ concentration ([Ca 2+ ] c ). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca 2+ ] c require Ca 2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca 2+ channels amplifying Ca 2+ release from lysosomes. GPN increases [Ca 2+ ] c by increasing pH cyt , which then directly stimulates Ca 2+ release from the ER. We conclude that physiologically relevant increases in pH cyt stimulate Ca 2+ release from the ER in a manner that is independent of IP 3 and ryanodine receptors, and that GPN does not selectively target lysosomes.

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