Dissecting the Molecular Mechanism of 2-APB-Induced Inhibition of Stim1-Orai1 Coupling

Abstract

Store-operated Ca2+ entry (SOCe) is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. SOCe is mediated by the ER Ca2+ sensing STIM proteins and the PM calcium channel, Orai. The aim of this study was to dissect the molecular mechanism of the inhibitory action of the SOCe modifying reagent, 2-aminoethoxydiphenyl borate (2-APB). We demonstrate that although 50 μM 2-APB can block SOCe, it does not prevent STIM1 puncta formation when Orai1 is co-expressed. Neither can it decrease STIM1-Orai1 FRET, thus the inhibitory effect of 2-APB is unlikely to be caused by any ability to block STIM1 puncta formation or physically uncouple STIM1 and Orai1. However, although preincubation of 2-APB does not inhibit Ca2+ influx through the action of the constitutively active Orai1-V102C in the absence of expressed STIM1, it can inhibit SOCe mediated by the combination of STIM1 and Orai1-V102C. Therefore, 2-APB preincubation can inhibit SOCe by functionally uncoupling STIM1 and Orai1. We identified one critical residue on STIM1 and several critical residues on the N-terminus of Orai1, both of which could affect functional STIM1-Orai1 coupling and SOCe in a graded manner. To determine possible inhibitory sites of 2-APB, we examined the effect of 2-APB incubation on SOCe mediated by these mutants and other reported mutations that could affect functional coupling between STIM1 and Orai1. Surprisingly, none of these mutations prevent the inhibition caused by 2-APB preincubation, indicating more critical residues are needed for functional STIM1-Orai1 coupling. Interestingly, one single mutation in the pore of Orai1, Orai1V102C, abolishes the ability of 2-APB to inhibit SOCe acutely. Thus, 2-APB may inhibit SOCe through its actions on the pore region of Orai1 channels.