Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca²⁺) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca²⁺. Store-operated Ca²⁺ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca²⁺ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca²⁺ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca²⁺ release from internal sources. The inefficacy of inhibitors of ER Ca²⁺ channels suggested that this compartment was not the source of the Ca²⁺ surge caused by tBHP exposure. However, using a Ca²⁺-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca²⁺ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca²⁺ homeostasis, with inhibition of mitochondrial Ca²⁺ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.