Orai-Stim Mediated Ca2+ Release from Secretory Granules Revealed by a Novel Ca2+ and pH Probe

Abstract

Secretory granules (SGs) bud from the trans-Golgi network and during subsequent maturation, acquire an acidic intraluminal pH, electron-dense cores, and matrices that bind Ca2+ and other ions. Total Ca2+ in SGs is remarkably high, 30-40 mM. The free Ca2+ is significantly lower, with estimates ranging from 10-80 μM. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty in measuring SG calcium directly in living cells. We adapted the FRET-based D1-ER probe to develop a novel probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca2+ of 69 ± 15 μM and pH 5.8. Application of ATP resulted in a slow monotonic decrease in SG Ca2+ temporally correlated with the occurrence of store-operated calcium-entry (SOCE). Further investigation revealed a novel receptor-mediated mechanism of calcium release from SGs that involves store-operated channels. SG Ca2+ release is completely antagonized by a SOCE antagonist, by switching to Ca2+-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca2+ by ∼75% and completely abolished the ATP-mediated release of Ca2+ from SG's. Overexpression of a dominant negative CAD construct (CAD-A376K) induced no significant changes in SG Ca2+. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels, and during SG Ca2+ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SGs membranes as a potential new mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the endoplasmic reticulum and exocytosis. (Grants NS08174/GM83913/F32 DC068982/K01 MH086119/NSFChina/985DEChina).