2-APB Induces Conformational Changes In STIM1 Cytoplasmic Domains To Couple And Activate CRAC Channel

Store-operated calcium channels?SOCs?are the major route of Ca²⁺entry in non-excitable cell,and play key roles in the control of gene expression,cell growth and differentiation,motility,secretion,tissue and organ development,and the immune response.Ca²⁺release-activated Ca²⁺?CRAC?channel is the most typical representative of SOCs.In resting cells replete with Ca²⁺,stromal interaction molecule1?STIM1?is diffusely localized throughout the endoplasmic reticulum?ER?in an inactive state;however,after ER Ca²⁺depletion,Ca²⁺release from the luminal Ca²⁺-binding EF-hand triggers unfolding of the EF-sterile?motif?SAM?domain,leading to conformational changes in both the luminal and cytosolic domains of STIM1 that trap STIM1 at junctionswhere it can interact with Calcium release-activated calcium channel protein 1?Orai1?N and C termini to induce store-operated Ca²⁺entry?SOCE?.The process of SOC activation offers many potential targets for pharmacological regulation of channel activity.2-Aminoethoxy-diphenyl borate?2-APB?is a widely used SOCE inhibitor and a powerful tool in the study of SOC.Previous studies have shown that 2-APB exerts a dose-dependent bimodal effect on SOCE,with strong enhancement of SOCE at low concentrations?1–10?M?and transient enhancement followed by complete inhibition at high concentrations?>20?M?.However,the mechanism by which 2-APB induces the coupling of STIM and Orai1 to activate CRAC is unclear.To investigate the mechanism of action of 2-APB on the C terminus of STIM1 in transient Orai1 activation,we designed a set of STIM1-derived FRET sensors containing different cytosolic domains of STIM1 to assess conformational changes within the STIM1 C terminus in the presence of 2-APB.We found that 2-APB reduced FRET in cells expressing double-labeled STIM1 C terminus?a.a.235-685,CT?and Orai1-activating small fragment?a.a.233-474,OASF??YFP-CT-CFP and YFP-OASF-CFP,respectively?,thereby promoting STIM1 C terminus–Orai1coupling and activating CRACs.At the same time we also found that Orai1 C terminal binding domain,?a.a.,272-292,CBD?rather than Orai1 N terminal binding domain,?a.a.73-85,NBD?play an important role in 2-APB induced STIM1 C terminus–Orai1 coupling,although both domains were required for 2-APB-mediated CRAC activation.We propose that 2-APB interferes with interactions that allosterically stabilize the coiled-coil?CC?1–CRAC activation domain?a.a.342-448,CAD?interface in the resting STIM1 C terminus to release the CAD which mediate interaction with CBD?and NBD?and CRAC current activation.We also found that the mechanism of CRAC activation by 2APB is consistent with the current model for SOC activation triggered by ER calcium depletion.