Authentic CRAC channel activity requires STIM1 and the conserved portion of the Orai N terminus

Calcium (Ca²⁺) is an essential second messenger required for diverse signaling processes in immune cells. Ca²⁺ release-activated Ca²⁺ (CRAC) channels represent one main Ca²⁺ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor in the endoplasmic reticulum, and the Ca²⁺ ion channel Orai in the plasma membrane. After Ca²⁺ store depletion, STIM1 and Orai couple to each other, allowing Ca²⁺ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca²⁺ selectivity, an increase in current density when switching from a Ca²⁺-containing solution to a divalent-free Na⁺ one, and fast Ca²⁺-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.