The ryanodine receptor agonist 4‐chloro‐3‐ethylphenol blocks ORAI store‐operated channels

Abstract

BACKGROUNDDepletion of the Ca 2+ store by ryanodine receptor (RyR) agonists induces store-operated Ca 2+ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE. EXPERIMENTAL APPROACHSOCE and ORAI1-3 currents were recorded by Ca 2+ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3. KEY RESULTS4-CEP induced a significant release of Ca 2+ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca 2+ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca 2+ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3. CONCLUSION AND IMPLICATIONS4-CEP not only acts as a RyR agonist to deplete the Ca 2+ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists. Abbreviations

Ethanolic Extract of Folium Sennae Mediates the Glucose Uptake of L6 Cells by GLUT4 and Ca2+

“…We found that 2-APB had no effect on FSE-mediated GLUT4 translocation under 2 mM extracellular Ca 2+ , but it evidently inhibited FSE-triggered Ca 2+ release ( Figure 6 A). The ryanodine receptor (RyR), another channel that releases Ca 2+ in the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) [ 40 ], has attracted much attention due to its manipulation of intracellular Ca 2+ output. Inhibition of RyR with 30 μM ryanodine had no effect on either GLUT4 translocation or intracellular Ca 2+ increase mediated by FSE ( Figure 6 B).…”

Multi-walled carbon nanotubes act as a chemokine and recruit macrophages by activating the PLC/IP3/CRAC channel signaling pathway

“…43 . T-REx HEK293 cells (Invitrogen) were routinely cultured and transfected with the above two plasmids to obtain stable expression of Orai1 and STIM1 in this cell line.…”

Functional validation and expression analysis of myotubes converted from skin fibroblasts using a simple direct reprogramming strategy

“…To assess intracellular Ca 2 + release, we studied the responses to chlorophenol derivatives and ATP. Ad.CAGMiC -infected human fibroblasts at day 14 or normal human fibroblasts were examined in the absence and presence of the ryanodine receptor stimulants 4-CEP or 4-CmC [16] . Extracellular ATP stimulates P2Y receptors to activate phospholipase C, thereby promoting the production of IP3 and the consequent stimulation of the IP3 receptor [17] .…”