Organellar Ca2+ shuttling and store-operated Ca2+ entry during nutrient starvation

Study Objective and Hypothesis: Macroautophagy is a process in which unwanted cellular components are packaged and delivered to the lysosome for degradation to maintain homeostasis. It is strongly activated by nutrient depletion (starvation), which induces an increase in intracellular Ca2+ (starvation-induced Ca2+ signal, SICS) that is predicted to initiate many Ca2+-dependent activities of autophagy. Despite the importance of SICS, its regulation is unclear. We hypothesized that the endoplasmic reticulum (ER) and the lysosome are linked sources of starvation-induced Ca2+ release, leading to store-operated Ca2+ entry (SOCE). We also hypothesized and that the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel is involved in the release of Ca2+ during starvation. Methods: SICR and SICS were measured by monitoring changes in intracellular Ca2+ concentration with the fura-2 Ca2+ indicator upon removal of extracellular nutrients in the absence or presence, respectively, of extracellular Ca2+. The roles of the ER, lysosome, and TRPML1 as components of SICR were assessed using pharmacological inhibitors and activators of the SERCA pump and TRPML1. Role of SOCE in SICS was tested with pharmacological inhibition or molecular truncation of the stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that is critical component of SOCE activation. Results: TRPML1 activation using agonist MLSA1 in the absence of extracellular Ca2+ triggered a small Ca2+ release signal; this effect is abolished by pre-depletion of ER Ca2+, indicating that the main source of lysosomal Ca2+ release via TRPML1 is ER Ca2+. Following starvation-induced Ca2+ release (SICR), TRPML1 agonist MLSA1 no longer triggers a Ca2+ release signal, indicating that lysosomal Ca2+ is a contributing source of SICR. Consistently, overexpression of TRPML1 enhances the kinetics and amplitude of SICS in the presence of extracellular Ca2+. Heterologous expression of STIM1, the critical molecular switcher of SOCE, increases SICS magnitude by 5-fold. This enhancement is abolished by expression of a STIM1 in which the intra-ER Ca2+-binding loop has been truncated. SICS is also strongly inhibited by pretreatment with STIM1 inhibitor ML-9. Conclusion: Our data support a model in which the ER and lysosome are linked sources of Ca2+ release during nutrient starvation, whose depletion leads to activation of store-operated Ca2+ entry. The data also suggest that TRPML1-mediated Ca2+ release contributes to the dynamics of Ca2+ release upon nutrient starvation. NIH grant HL173818 to Q-KT This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.