Michelle Yen

Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel

Store-operated Ca2+ entry occurs through the binding of the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca2+-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca2+ block, La3+ sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca2+ affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

Orai1 pore residues control CRAC channel inactivation independently of calmodulin.

Researchers have reevaluated the role of calmodulin and previously identified calmodulin binding sites in the mechanism by which Ca2+-release activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

Correction: Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Volume 147, No. 2, February 1, 2016. Pages [137–152][1]. The Journal of General Physiology regrets mistakes that appeared in the original publication of this paper, as the result of production errors. The equation in the first paragraph of Materials and methods section Data analysis did not

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.