The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels |
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Authors: | Bo Zeng Gui-Lan Chen Nikoleta Daskoulidou Shang-Zhong Xu |
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Affiliation: | 1.Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, UK;2.Key Laboratory for Medical Electrophysiology, Ministry of Education of China, and the Institute of Cardiovascular Research, Luzhou Medical College, Luzhou, China |
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Abstract: | BACKGROUNDDepletion of the Ca2+ store by ryanodine receptor (RyR) agonists induces store-operated Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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. |
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Keywords: | ryanodine receptors store-operated Ca2+ channels STIM1 ORAI 4-chloro-3-ethylphenol 4-chloro-m-cresol |
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