Roles of the Orai1 C-Terminus and N-Terminus in 2-APB-Induced STIM1 Coupling

Abstract

The small molecule, 2-aminoethoxydiphenyl borane (2-APB) has complex effects on store-operated Ca2+ entry (SOCE). Higher levels (50 μM) strongly inhibit STIM1-miedated Orai1 channel activation. However, using the poorly active STIM1 C-terminal domain (S1CT) 235-685), 50 μM 2-APB greatly enhances binding to and activation of Orai1 channels. We assessed which parts of the Orai1 channel were required for this coupling interaction. Using the Orai1 C-terminal truncation of Orai1 (removal of residues 267-301), 50 μM 2-APB caused neither binding between S1CT and truncated Orai1 nor Ca2+ entry. We replaced the missing Orai1 C-terminus with the FKBP-rapamycin binding (FRB) domain of the mTOR protein, and added the 12-kDa FK506- and rapamycin-binding protein (FKBP12) to the carboxyl end of S1CT. There was no Ca2+ entry even when S1CT and truncated Orai1(1-266) were physically tethered through rapamycin-induced FRB/FKBP12 interactions. However, the addition of 2-APB now recovered full Ca2+ entry. Thus, the C-terminus of Orai1 (267-301) is not specifically required and can be substituted by a simple binding interaction between FRB/FKBP12. We also revealed using FRET and fluorescence imaging, that the 267-301 C-terminus of Orai1 attached to the CFP-tagged PM-localization construct, was alone sufficient to trap S1CT upon the addition of 50 μM 2-APB. Finally, we identified a single residue (Leu-79) in the N-terminus of Orai1 that is critical for the effects of 2-APB on SOCE. The L79N Orai1 mutant completely abolished the activation effect of 50 μM 2-APB on S1CT, but only partially prevented the action of store-induced STIM1 activation of Orai1. Our data provide new insights into our understanding of 2-APP-induced activation of STIM1/Orai1 coupling.