2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this is...2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this issue,we systematically employed confocal imaging,dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orail,two key components of CRACs.Imaging results support that there are two signaling pathways(Orail-independent and Orail-dependent)for the formation of STM1 puncta.2 APB could dose dependently block Orail-independent but not Oril-dependent STIM1 puncta formation,despite its obvious inhibition effect on store-opented Ca^(2+)entry(SOCE).In addition,we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization,it could completely block CAD-YFP-induced constitutive Ca^(2+)entry and promnote the interaction between Orail and CAD by FRET mea-surements.Therefore,we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhbitory effects on Orail channel itself,but not the interference on puncta formation between STIM1 and Orail.展开更多
基金supported by the National Natural Sciences Foundation of China(Grant Nos.31371217 and 30871311).
文摘2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this issue,we systematically employed confocal imaging,dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orail,two key components of CRACs.Imaging results support that there are two signaling pathways(Orail-independent and Orail-dependent)for the formation of STM1 puncta.2 APB could dose dependently block Orail-independent but not Oril-dependent STIM1 puncta formation,despite its obvious inhibition effect on store-opented Ca^(2+)entry(SOCE).In addition,we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization,it could completely block CAD-YFP-induced constitutive Ca^(2+)entry and promnote the interaction between Orail and CAD by FRET mea-surements.Therefore,we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhbitory effects on Orail channel itself,but not the interference on puncta formation between STIM1 and Orail.
