Abstract
ABSTRACTPharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca2+ entry (SOCE) by these agents. Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes (bafilomycin A1, lysosomal re-positioning) had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.
Highlights
Membrane-permeant lysosomotropic agents have long been used to disrupt lysosomal function and include two distinct classes: peptides or cationic amphiphilic drugs
R-CEPIA1er showed antiparallel changes in [Ca2+]endoplasmic reticulum (ER) compared with the cytosolic Ca2+ concentration ([Ca2+]cyt) as simultaneously monitored with GCaMP6s: cyclopiazonic acid (CPA) and ionomycin induced a complete emptying of the ER and an accompanying high cytosolic Ca2+ plateau (Fig. 1A-C)
Turning to the effect of lysosomal agents, we found that both glycylL-phenylalanine 2-naphthylamide (GPN) and LLOMe profoundly inhibited hBACCS2-dependent responses (Fig. 6J-L); GPN and LLOMe both promoted a decrease in Ca2+ that was faster than that seen upon the removal of activating light, but not as fast as the effect of EGTA (Fig. 6M)
Summary
Membrane-permeant lysosomotropic agents have long been used to disrupt lysosomal function and include two distinct classes: peptides or cationic amphiphilic drugs. Peptides such as glycylL-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester (LLOMe) are thought to be cleaved by lysosomal proteases and result in lysosomal osmotic rupture and lysosomal Ca2+ release (Morgan et al, 2020). Lysosomotropic agents can evoke a compromise of the lysosomal membrane integrity, referred to as lysosomal membrane permeabilization (LMP) (Villamil Giraldo et al, 2014; Wang et al, 2018). Regardless of the precise mechanism, it is agreed that lysosomotropic agents evoke Ca2+ release from the endoplasmic reticulum (ER) Ca2+ stores (Morgan et al, 2020), it is just unclear whether this is secondary to an action
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