Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

Abstract

The Ca 2؉ release-activated Ca 2؉ channel is a principal regulator of intracellular Ca 2؉ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca 2؉ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca 2؉ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150 Å in height and 95 Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100 Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.

A Microscopic View of the Store-Operated Calcium Entry-Pathway

“…Furthermore, these results were confirmed by conducting FRET experiments between covalently linked Orai1 homodimers CFP/YFP-labeled, resulting in a significant interaction between homodimers, regardless of whether STIM1 is cotransfected or ER was depleted [24]. Maruyama et al confirmed the same results through purified Orai1 FLAG tagged in reconstructed three-dimensional structures from negatively stained electron microscopy images with a resolution of 21Å [20]. By using 3681 images from purified Orai1, they resolved a teardrop-shaped channel in its basal conformation, with 4-fold symmetry from its top view and with the largest portion of the protein facing the cytoplasm.…”

“…However, there is vast microscopy evidence from several groups reporting a tetrameric configuration, both in functional and resting channels [20,[22][23][24], by using single molecule imaging technique, with total internal reflection fluorescence Microscopy (TIRFM) in HEK293 [22] and Xenopus oocytes [21]. Through the use of molecular constructs of Orai1 fused to the green fluorescent protein (GFP) and continued exposure to strong laser excitation, bleaching steps of the fluorescent protein were counted in order to estimate the number of subunits in single molecule studies of the channel [21,22,25].…”

“…Dimers in resting state, tetramers, hexamers, and even high molecular aggregates are common results in assays where concentration of the cross-linking agent is directly proportional to the grade of oligomerization [18,20,21].…”

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

“…We and others have previously demonstrated that Orai1 forms tetramers in the PM [17][18][19][20]. We have also demonstrated that one CRAC channel contains at least two STIM1 full-length cytosolic fragments (CT-STIM1) [17][18][19][20], which suggests that a STIM1 dimer may be enough to open the CRAC channel. This result seems to disagree with the general belief that the oligomerization of full-length STIM1 is required for the channel activation [21,22].…”

Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91

“…Wild-type Orai1 proteins were detected primarily as a monomer band, consistent with its predicted molecular weight (∼34 kDa) with or without diamide treatment. We note that even without diamide treatment, there was some residual level of Myc-Orai1 oligomers from both wildtype and R91C mutants, most likely due to the higher-order stoichiometry of Orai1 as membrane proteins with dimer or tetramer structure in intact cells (18,20,29,30), along with incomplete dissociation during cell-lysate treatment. However, for R91C, diamide treatment caused the monomer band to disappear as a strong band at higher molecular weight emerged, consistent with covalent dimer formation.…”