Stoichiometry of CRAC Channel Assembly and Gating

CRAC channels are opened by binding of the ER calcium sensor STIM1 to the C-terminus of the channel subunit Orai1. Previous functional experiments suggested a tetrameric channel stoichiometry, but the crystal structure of Drosophila Orai is a trimer of dimers, with each C-terminus forming a coiled-coil with its neighbor. This raises two fundamental questions: what is the stoichiometry of the CRAC channel, and does STIM1 bind to individual or pairs of C-termini to open it? To address these questions, we constructed hexameric concatemers of Orai1. Orai1 hexamers produced currents with properties that were indistinguishable from native ICRAC, including Ca2+ selectivity, Ca2+-dependent inactivation, and modulation by 2-APB. The inhibitory effects of single L273D mutations confirmed that all 6 subunits participated equally in forming the functional channel.STIM1-Orai1 binding was studied using E-FRET between STIM1-YFP and CFP-Orai1. While the Orai1(L273D) C-terminus alone did not bind STIM1, it enhanced binding when paired with a neighboring WT C-terminus. To compare how monomer vs dimer binding are coupled to channel opening, we constructed hexamers with a single truncated or L273D C-terminus. The truncated hexamer showed significantly less activity than the L273D mutant, arguing against a pure monomeric gating mode.The relationship between STIM1 occupancy and channel activation was examined using Orai1 hexamers containing 1-3 STIM-binding mutations (producing 1-3 Orai1 heterodimers per channel). For both strong (L273D) and weak (L286S) inhibitory mutations, channel activity was well described by a model that assumes independent and equal energetic contributions from each heterodimer.In summary, we present the first functional evidence that hexameric Orai1 channels have the same properties as native CRAC channels. Our data suggest that STIM1 binds pairs of Orai C-termini and opens CRAC channels as a trimer of dimers, with each dimer contributing a constant amount of gating energy.