Distances and charges along the Orai1 nexus-TM3 interface control STIM1-binding and pore opening

Calcium (Ca ²⁺ ) influx through the Ca ²⁺ release-activated Ca ²⁺ (CRAC) channel is triggered by binding of the Ca ²⁺ sensor Stromal Interaction Molecule 1 (STIM1) to the pore-forming Orai1 complex, primarily to its cytosolic C-termini. These C-termini connect to the transmembrane domain (TM) 4 via the flexible nexus region, proposed to transmit the activation signal from the STIM1-binding site to the central pore via concentrically arranged TM domains. However, the conformational dynamics of the nexus-TM3 interface required for channel gating remain elusive. Here, we investigate its role using unnatural amino acid (UAA)-based photo- and chemical crosslinking at individual positions within the nexus-TM3 interface combined with conventional site-directed mutagenesis. We report that a widening of the nexus-TM3 interface is essential for STIM1-mediated pore opening, while hydrophobicity and contact distances in the upper nexus-TM3 interface fine-tune signal propagation to the pore. These findings underscore the relevance of the nexus-TM3 dynamics for proper Orai1 function.