The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca 2+ 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 Ca 2+ 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.

The inactivation domain of STIM1 (ID STIM : amino acids 470–491) has been described as necessary for Ca 2+ -dependent inactivation (CDI) of Ca 2+ release–activated Ca 2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID STIM that control the extent of CDI and examine functional interactions of ID STIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three ID STIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling ID STIM by a triple alanine substitution for these three residues (“STIM1 3A”) or by truncation of the entire domain (STIM1 1–469 ) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM1 1–469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca 2+ conductance, factors that determine local Ca 2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between ID STIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling ID STIM or W76 were not additive, demonstrating that ID STIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling ID STIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking ID STIM generated approximately two thirds of the WT extent of CDI but those with ID STIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although ID STIM is not required as a Ca 2+ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.

Ca 2+ entry through CRAC channels causes fast Ca 2+ -dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca 2+ –CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl − modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

CRAC (calcium release-activated Ca 2+ ) channels attain an extremely high selectivity for Ca 2+ from the blockade of monovalent cation permeation by Ca 2+ within the pore. In this study we have exploited the blockade by Ca 2+ to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 Å. Extracellular Ca 2+ blocks monovalent flux in a manner consistent with a single intrapore site having an effective K i of 20 μM at −110 mV. Block increases with hyperpolarization, but declines below −100 mV, most likely due to permeation of Ca 2+ . Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca 2+ indicates an open probability ( P o ) of ∼0.8. By extrapolating the variance/mean current ratio to the condition of full blockade ( P o = 0), we estimate a unitary conductance of ∼0.7 pS for Na + , or three to fourfold higher than previous estimates. Removal of extracellular Ca 2+ causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance I CRAC increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca 2+ , and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.

Although store-operated calcium release–activated Ca 2+ (CRAC) channels are highly Ca 2+ -selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na + and Cs + with a unitary conductance of ∼40 pS, and that intracellular Mg 2+ modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg 2+ -inhibited cation (MIC) channels that open as Mg 2+ is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg 2+ to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca 2+ to divalent-free extracellular solution evokes Na + current through open CRAC channels (Na + -I CRAC ) that is initially eightfold larger than the preceding Ca 2+ current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs + (P Cs /P Na = 0.13 vs. 1.2 for MIC). Neither the decline in Na + -I CRAC nor its low Cs + permeability are affected by intracellular Mg 2+ (90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca 2+ channels.