title: The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.
abstract: BACKGROUND
  Depletion 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.
  
  
  METHODS
  SOCE 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.
  
  
  RESULTS
  4-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.
  
  
  CONCLUSIONS
  4-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.
authors.affiliation: Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, UK.
authors.forename: Bo
authors.initials: B
authors.lastname: Zeng
authors.forename: Gui-Lan
authors.initials: GL
authors.lastname: Chen
authors.forename: Nikoleta
authors.initials: N
authors.lastname: Daskoulidou
authors.forename: Shang-Zhong
authors.initials: SZ
authors.lastname: Xu
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002733:Chlorophenols / Q000494:pharmacology*; D003408:Cresols / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D000071740:ORAI1 Protein; D000071741:ORAI2 Protein; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1
chemical_list: D002120:Calcium Channel Agonists; D015220:Calcium Channels; D002733:Chlorophenols; D003408:Cresols; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D000071741:ORAI2 Protein; C514506:ORAI2 protein, human; C522867:Orai3 protein, human; D019837:Ryanodine Receptor Calcium Release Channel; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; C091710:4-chloro-3-ethylphenol; C006984:chlorocresol; C029107:4-chlorophenol; D002118:Calcium
keywords: 4-chloro-3-ethylphenol; 4-chloro-m-cresol; ORAI; STIM1; ryanodine receptors; store-operated Ca2+ channels