The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by abrogating STIM1/Orai1 interaction

Abstract

The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca2+ entry (SOCE), a ubiquitous Ca2+ entry pathway regulating multiple cellular functions, is unknown. Here we show that NAGly but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic β-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca2+ depletion of the endoplasmic reticulum (ER), while it was not effective on Ca2+ entry via L-type voltage-gated Ca2+ channels. Enhanced Ca2+ entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, the stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly neither affected STIM1 oligomerization, nor STIM1 clustering, nor the co-localization of STIM1 with Orai1, which were induced by Ca2+ depletion of the ER. In contrast, independently from its slow depolarizing effect on mitochondria NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings unveiled the STIM1/Orai1-mediated SOCE machinery as a so far unknown molecular target of NAGly, which might have multiple implications in cell physiology.