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

Calcium (Ca²⁺) influx through the Ca²⁺ release-activated Ca²⁺ (CRAC) channel is triggered by binding of the Ca²⁺ sensor STIM1 to the pore-forming Orai1 complex, primarily to its cytosolic C termini. These C termini connect to transmembrane (TM) domain 4 via the flexible nexus region, which has been proposed to transmit the activation signal toward 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 combine unnatural amino acid (UAA)-based photocrosslinking and chemical crosslinking with site-directed mutagenesis to investigate this interface. We report that a widening of the nexus-TM3 interface is an essential step within the cascade of conformational rearrangements underlying STIM1-mediated pore opening, while hydrophobicity and charges in this interface are further determinants contributing to signal propagation to the pore. These findings underscore the relevance of the nexus-TM3 dynamics for proper Orai1 function.