Probing the Functional Coupling Interface between STIM1 and Orai1

Abstract

Store operated Ca2+ entry (SOCE) plays a crucial physiological roles and involves two key proteins, STIM and Orai. Triggered by store-depletion, ER STIM proteins aggregate at ER-PM junctions and directly activate PM Orai Ca2+ channels. Few SOCE inhibitors have been described that specifically target the STIM/Orai machinery. Recently, we revealed a previously described SOCE modifier, DPB162-AE, potently and specifically blocks STIM1-induced Orai1 channel activation. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not affect SOAR-dimerization nor does it prevent the SOAR-Orai1 interaction. However, it potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores Orai-binding of this SOAR mutant but only slowly restores its activation of Orai1 channel-mediated Ca2+ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. In STIM2, the equivalent residue to Phe-394 is Leu-485 resulting in STIM2's lower efficacy for Orai channel activation. This difference may explain the unusual specificity of DPB162-AE in blocking the action of STIM1 but not STIM2. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.