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 the transmembrane domain (TM) 4 via the flexible nexus region, proposed to transmit the activation signal towards 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 combined unnatural amino acids (UAA)-based photo- and chemical crosslinking with site-directed mutagenesis to investigate this interface. We report that a widening of the lower nexus-TM3 interface is an essential step within the cascade of conformational rearrangements underlying STIM1-mediated pore opening, while hydrophobicity and contact distances in the upper nexus-TM3 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.