The Mechanism Of STIM1 Regulating Orai1 Channel By 2-APB

Storage-operated calcium entry?SOCE?is a kind of external calcium influx induced by the decrease or depletion of calcium concentration in endoplasmic reticulum?ER?.The ion channel that mediates SOCE is called store-operated calcium channels?SOCs?.Calcium release-activated Ca²⁺channel?CRAC?is the most typical type of SOCs.Its main components are stromal interaction molecule protein?STIM?and calcium release-activated calcium channel protein?Orai?,also known as calcium release-activated calcium channel regulatory factor?CRACM?.Orai family has three homologous proteins:Orai1,Orai2 and Orai3;STIM has two subtypes:STIM1 and STIM2.Classical SOCE is mediated by STIM1 and Orai1.STIM1 in the endoplasmic reticulum is a single transmembrane protein:the N-terminal of the endoplasmic reticulum acts as a receptor to sense the change of calcium concentration in the calcium pool;the TM region of the endoplasmic reticulum transmits the signal of the change of calcium concentration in the endoplasmic reticulum cavity to the free C-terminal of the cytoplasm;the C-terminal of the cytoplasm forms oligomers and translocates to the position of the endoplasmic reticulum near the cell membrane to form a puncta structure,which binds and activates Orai1 channel.Orai1,a four-transmembrane channel protein on the cell membrane,is a highly selective Ca²⁺channel with both N and C-terminals in the cell.Orai1 usually exists in the form of dimer,which is activated by STIM1 to form a channel-active hexamer,triggering a small sustained calcium influx and maintaining cell calcium homeostasis.This inward calcium current is called calcium release-activated Ca²⁺current(I_CRAC).The function of CRAC channel is closely related to the immune function of organism.It directly regulates the physiological functions of many immune cells,such as T cells,B cells and mast cells,to participate in the immune response.The mutation of CRAC channel gene leads to functional deficiency,which can lead to dystonia,autoimmune disease,dyshidrosis,ectodermal dysplasia and hemorrhagic diseases,and even severe combined immunodeficiency?SCID?,which endangers life safety.Although the molecular structure of CRAC channel has been revealed,the molecular mechanism of CRAC channel regulation still needs to be further studied.2-Aminoethoxy-diphenyl borate?2-APB?is a membrane permeability compound,which has been proved to be an inhibitor of inositol triphosphate?IP₃R?receptor in the early stage.Later studies found that 2-APB can effectively inhibit SOCE as a non-specific channel blocker,and has become a common tool for the study of CRAC channels.The effects of 2-APB on CRAC channels are very complex:2-APB at low concentration?<5?M?enhances STIM1-activated ICRAC;2-APB at high concentration?50?M?exhibits rapid inhibition of STIM1-activated ICRAC after transient enhancement;2-APB at high concentration?50?M?directly gates Orai3 and Orai1 without STIM1.The molecular mechanism of 2-APB activating CRAC channel is still unclear.Some people think that 2-APB activation of CRAC does not require STIM1,but directly acts on activated Orai1,expanding the pore diameter and promoting calcium influx.However,this study found that 2-APB can activate Orai1by exposing the CAD domain by altering the CT-terminal conformation of STIM1 to produce calcium influx.1.2-APB Induces Interaction between STIM1 CT-terminal and Orai1 In order to study the molecular mechanism of 2-APB enhancing CRAC current,YFP-CT-CFP was co-transfected into Hela cells by STIM1 CT-terminal?a.a.235-685,C-terminal,CT?and Orai1-mKate.After 2-APB applied,YFP-CT-CFP,which was uniformly distributed in cytoplasm,was translocated to the cell membrane and co-localized with Orai1-mKate.It was suggested that 2-APB can induce YFP-CT-CFP and Orai1-mKate to form complex.YFP-CT-CFP and Orai1-YFP were co-transfected into Hela cells.Fluorescence resonance energy transfer?FRET?was used to monitor the distance between the two proteins.Under the excitation light of fluorescent label protein CFP with a wavelength of 405 nm,signals were collected at the emission wavelengths of CFP and YFP respectively and stimulated by 2-APB.Laser scanning confocal microscopy showed that YFP-CT-CFP with uniform distribution in cytoplasm was translocated to the co-localization of cell membrane and Orai1-YFP,and the FRET between the two molecules increased rapidly.The molecular distance between STIM1 CT-terminal carrying CFP and Orai1 carrying YFP is less than 10 nm,that is,there is a direct interaction between them.These results suggest that 2-APB induces interaction between STIM1 CT-terminal and Orai1.2.2-APB induces interaction between STIM1 CT-terminal and Orai1 and activates Orai1 channel CMV-R-GECO1.2,a red genetically-encoded Ca²⁺indicator?GECI?,was used to monitor intracellular Ca²⁺concentration.YFP-CT-CFP,Orai1-CFP and CMV-R-GECO1.2 were transfected into Hela cells,and 2-APB with different concentrations were added.Laser scanning confocal microscopy showed that low concentration of 2-APB?<5?M?induced persistently activated Ca²⁺entry,while high concentration?>20?M?activated strong Ca²⁺influx first and then quickly blocked Ca²⁺influx.3.2-APB Induces conformational transition of the CAD domain of STIM1CT-terminal In order to study how to couple Orai1 within STIM1 CT-terminal,YFP-CT-CFP was first transfected into Hela cells and stimulated with 2-APB.Confocal microscopy recorded the decrease of FRET efficiency,suggesting that the folded structure of STIM1 CT-terminal can be transformed into a straight conformation.Then the same double fluorescent label protein?YFP-CAD-CFP?was linked to the CRAC activation domain?CAD?of STIM1 CT-terminal.After the constructed plasmid was transferred into Hela cells,the above steps were repeated.Confocal observation showed that although the efficiency of FRET decreased,there was no plaque aggregation and no translocation to the cell membrane.When co-rotating with Orai1-mKate,there was no co-localization between CAD and overexpressed Orai1.It is revealed that 2-APB can also induce conformational transition of the CAD domain of STIM1 CT-terminal.4.2-APB-induced interaction between STIM1 CT terminal and Orai1depends on the CBD and NBD domains of Orai1 In this study,we used Orai1-?CBD-mKate which was knocked out STIM1 binding domain?a.a.272-292,C-terminal binding domain,CBD?at the C-terminal regions of Orai1,Orai1-?NBD-mKate which was knocked out STIM1 binding domain?a.a.73-85,N-terminal binding domain,NBD?at the N-terminal regions of Orai1 co-transfected with YFP-CT-CFP respectively.Hela cells were treated with 2-APB,and fluorescence changes were recorded by confocal recording.The results showed that there was no YFP-CT-CFP localization in Hela cells knocked out of CBD,while the absence of NBD only showed a decrease in localization.It was suggested that the interaction between Orai1 and STIM1 CT-terminal induced by 2-APB depends on the CBD fragment of Orai1,and NBD seems not to play a major role in this process.Conclusion:This study demonstrates that 2-APB can activate Orai1 by inducing conformational changes of STIM1 CT-terminal,exposing the Orai1 activation site,and inducing direct interaction between STIM1 CT-terminal and Orai1,thereby activating Orai1 and triggering intracellular calcium influx.CBD and NBD fragments of Orai1 were both associated with the binding of STIM1 CT-terminal to Orai1induced by 2-APB.