ABSTRACT
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 (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]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 [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.
Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Cytosol/drug effects , Dipeptides/pharmacology , Endoplasmic Reticulum/drug effects , Biological Transport , CRISPR-Cas Systems , Calcium Channels/genetics , Calcium Channels/metabolism , Cathepsin C/genetics , Cathepsin C/metabolism , Cell Line, Tumor , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Gene Editing , Gene Expression , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration/drug effects , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/metabolism , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Ploidies , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
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.
