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

Abstract

SummaryThe 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 Ca 2+ entry (SOCE), a ubiquitous Ca 2+ entry pathway regulating many 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 b-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca 2+ depletion of the endoplasmic reticulum, whereas it had no effect on Ca 2+ entry through L-type voltage-gated Ca 2+ channels. Enhanced Ca 2+ entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca 2+ depletion of the endoplasmic reticulum. 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 revealed the STIM1-Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.

N‐arachidonoyl glycine suppresses Na+/Ca2+ exchanger‐mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

“…Importantly, NAGly appears to have complex effects on different key ion transport systems expressed in endothelial cells. Recently we showed that in cells overexpressing the key molecular constituents of SOCE, the stromal interacting molecule 1 (STIM1) and the pore‐forming subunit of SOCE channels, Orai1, NAGly hampers SOCE primarily by uncoupling STIM1 from Orai1 (Deak et al ., ). Accordingly, that and our current data indicate that endogenous lipoaminoacid NAGly fine‐tunes the interplay between crucial molecular players of cellular signalling.…”

N‐Acyl amino acids and their impact on biological processes

“…Recently, Bondarenko et al reported a previously unrecognized inhibitory effect of NAGly on plasma membrane Na 1 -Ca 21 exchange activity, which largely controls endothelial cell function [34]. NAGly has also been shown to reversibly hinder store-operated Ca 21 entry (a ubiquitous Ca 21 entry pathway regulating many cellular functions) in a time-and concentration-dependent manner [35]. These data unveiled an unknown inhibitory effect of NAGly on the signaling between stromal interacting molecule 1 (STIM1), a major contributor to Ca 21 signaling by means of its activity as Ca 21 channel regulator and Orai1 (a Ca 21 -selective ion pore).…”

Endothelial atypical cannabinoid receptor: do we have enough evidence?

“…In a GPCR‐independent manner, anandamide and NAGly inhibit both forward and reverse modes of Na + ‐Ca 2+ exchanger in endothelial cells (Bondarenko et al ., ). In addition, NAGly was shown to directly potentiate the BK Ca channels (Bondarenko et al ., ) and inhibit store‐operated Ca 2+ entry in different cell types (Deak et al ., ). Because vascular signalling and function are chiefly regulated by Na + ‐Ca 2+ exchanger (Bondarenko, ; Andrikopoulos et al ., ), IK Ca , BK Ca (Vang et al ., ; Bondarenko et al ., ; Wulff and Kohler, ) and different members of TRP channels, identification of their contribution to GPCR‐dependent and ‐independent cannabinoid signalling still needs to be clarified.…”

“…However, in rat small mesenteric arteries, NAGly evoked a concentration‐dependent relaxation with an EC 50 value of 5.8 μM (Parmar and Ho, ), which is several orders higher than that required to activate GPR18. At these concentrations, NAGly exhibits a direct GPCR‐independent effect on a number of ion transport systems expressed in both endothelial and smooth muscle cells (Bondarenko et al ., ; Deak et al ., ). In endometrial cell line, HEC‐1B, THC was shown to be a full agonist for GPR18, with a calculated EC 50 value of 0.96 μM (McHugh et al ., ).…”