An effective get-together

The release of calcium (Ca²⁺) from the endoplasmic reticulum (ER) is followed by the influx of Ca²⁺ across the plasma membrane through Ca²⁺-release-activated Ca²⁺ (CRAC) channels, which are crucial for sustained Ca²⁺ signalling in many cell types. Three studies now provide new insights into how Ca²⁺ depletion is sensed in the ER and discover a unique mechanism for ion channel assembly and activation.

The activity of CRAC channels relies on the ER transmembrane protein stromal interaction molecule-1 (STIM1), which senses the depletion of Ca²⁺ in the ER and physically relays this information to the plasma membrane protein ORAI1, which forms the pore of the Ca²⁺ channel.

Using nuclear magnetic resonance (NMR), Stathopulos et al. determined the three-dimensional atomic structure of the Ca²⁺-sensing region of STIM1, which consists of the EF-hand and sterile α-motif (SAM) domains (EF–SAM domains). They found a 'hidden' EF-hand domain that stabilizes the EF–SAM intramolecular interaction. Mutations that alter the structure of the SAM, EF-hand or hidden EF-hand domains unfold the EF–SAM domain and disrupt Ca²⁺ sensitivity. Furthermore, they induce Ca²⁺ flux independently of the ER Ca²⁺ store. So, the conformation of the STIM1 Ca²⁺-sensing region is crucial for store-operated Ca²⁺ entry.

Penna et al. transfected haemagglutinin (HA)-tagged Drosophila melanogaster Orai into S2 cells and performed chemical crosslinking assays on intact cells and total cell lysates. They found that Orai forms homodimers in the plasma membrane of resting cells. Similar results were obtained in a human kidney cell line using gel electrophoresis under non-dissociating conditions.

Expression of the cytosolic C-terminal region of Stim (C-Stim), together with green fluorescent protein (GFP)-tagged Orai, showed colocalization of the two proteins and induced constitutive Ca²⁺ influx in S2 cells without causing Ca²⁺ store depletion. Furthermore, C-Stim induced homomultimeric association of Orai in biochemical assays. So, the oligomeric state of Orai depends on C-Stim binding and the C-Stim–Orai interaction is independent of the ER Ca²⁺ store content.

“...a new mechanism for ion channel activation...”

In an independent study, Ji et al. used single-molecule imaging and fluorescence resonance energy transfer (FRET) to study the assembly of functional CRAC channels, and found that four human ORAI1 subunits and two STIM1 molecules assemble to form the active channel. However, Ji et al. also found that ORAI1 formed tetramers in resting cells.

Penna et al. then investigated the mechanism that underlies the assembly and activation of CRAC channel. Imaging of single molecules of GFP–Orai that were expressed in Xenopus laevis oocytes by total internal reflection microscopy (TIRFM) and photobleaching confirmed the dimeric basal state of Orai that was initially observed in S2 cells. Furthermore, coexpression of C-Stim induced the assembly of GFP–Orai into tetramers that constitute the active CRAC channel.

Together, these data illustrate a new mechanism for ion channel activation, which requires an activator protein (STIM1) to assemble and open the ORAI1 tetrameric channel in the plasma membrane.