Molecular determinants of fast Ca²⁺-dependent inactivation and gating of the Orai channels

Ca²⁺ influx by store-operated Ca²⁺ influx channels (SOCs) mediates many cellular functions regulated by Ca²⁺, and excessive SOCmediated Ca²⁺ influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca²⁺-dependent inactivation, which limits Ca²⁺ influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca ²⁺-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca ²⁺-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca²⁺-dependent inactivation. Fast Ca²⁺-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca ²⁺-dependent inactivation that has implications for Ca²⁺ influx by SOC in physiological and pathological states.