Novel pancreatic cancer susceptibility loci discovered through genome-wide association studies

s / Pancreatology 15 (2015) S1eS141 S49 sensor protein Stim1 in response to Ca2þ release from the ER. Orai1then undergoes slow Ca2þ -dependent inactivation (SCDI) that is mediated by binding of the regulator protein SARAF to Stim1. Aims: In this study our aimwas to characterize the regulation of Orai1. Materials u0026 methods: Human embryonic kidney 293 cells were transfected with different plasmid constructs. We used SCDI by SARAF as a reporter of the conformation and microdomain localization of the Orai1Stim1 complex. FRET was used to follow interaction of Stim1-SARAF and Ca2þ current measurement to determine Orai1 activity. Results: Interaction of SARAF with Stim1 required the presence of Stim1-Orai1 in the ER/PMmicrodomain that is tethered by E-Syt1, stabilized by Septin4 and enriched in PI(4,5)P2. Notably, selective targeting of Stim1 to PI(4,5)P2-rich or to PI(4,5)P2-poor microdomains revealed that SCDI by SARAF is observed only when the Stim1-Orai1 complex is within the PI(4,5) P2-rich microdomain. Measuring the dynamics of Stim1-Orai1 localization using PI(4,5)P2-rich or PI(4,5)P2-poor microdomain probes revealed that store depletion is followed by transient Stim1-Orai1 formation in the PI(4,5) P2-poor microdomain where the channel is fully active, which then translocates to the PI(4,5)P2-rich domain to recruit SARAF and initiates SCDI. Conclusion: These findings reveal the role of the ER/PM tethers in the regulation of Orai1 function and Ca2þ influx and describe a new mode of regulation by PI(4,5)P2 involving translocation between PI(4,5)P2 microdomains.