The molecular determinants of gating and regulation of the calcium channel Orai1 by the ER Ca2+ sensor STIM1 during store-operated calcium entry are now defined. Influx of Ca2+ through store-operated Ca2+ channels (SOCs) is a central component of receptor-evoked Ca2+ signals1. Orai channels are SOCs2,3,4 that are gated by STIM1, a Ca2+ sensor located in the ER5,6 but how it gates and regulates the Orai channels is unknown. Here, we report the molecular basis for gating of Orais by STIM1. All Orai channels are fully activated by the conserved STIM1 amino acid fragment 344–442, which we termed SOAR (the STIM1 Orai activating region). SOAR acts in combination with STIM1 (450–485) to regulate the strength of interaction with Orai1. Activation of Orai1 by SOAR recapitulates all the kinetic properties of Orai1 activation by STIM1. However, mutations of STIM1 within SOAR prevent activation of Orai1 but not co-clustering of STIM1 and Orai1 in response to Ca2+ store depletion, indicating that STIM1–Orai1 co-clustering is not sufficient for Orai1 activation. An intact carboxy terminus α-helicial region of Orai is required for activation by SOAR. Deleting most of the Orai1 amino terminus impaired Orai1 activation by STIM1, but Orai1Δ1–73 interacted with and was fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1 (672–685) and a Pro-rich region in the N terminus of Orai1. Hence, the essential properties of Orai1 function can be rationalized by interactions with discrete regions of STIM1.

SOAR and the polybasic STIM1 domains gate and regulate Orai channels