CRAC Activation Domain Research Articles

Lead inhibits microglial cell migration via suppression of store-operated calcium entry

Lead (Pb) is a non-biodegradable environmental pollutant that can lead to neurotoxicity by inducing neuroinflammation. Microglial activation plays a key role in neuroinflammation, and microglial migration is one of its main features. However, whether Pb affects microglial migration has not yet been elucidated. Herein, the effect of Pb on microglial migration was investigated using BV-2 microglial cells and primary microglial cells. The results showed that cell activation markers (TNF-α and CD206) in BV-2 cells were increased after Pb treatment. The migration ability of microglia was inhibited by Pb. Both store-operated calcium entry (SOCE) and the Ca2+ release-activated Ca2+ (CRAC) current were downregulated by microglia treatment with Pb in a dose-dependent manner. However, there was no statistical difference in the protein levels of stromal interaction molecule (STIM) 1, STIM2, or Ca2+ release-activated Ca2+ channel protein (Orai) 1 in microglia. The external Ca2+ influx and cell migration ability were restored to a certain extent after overexpression of either STIM1 or its CRAC activation domain in microglia. These results indicated that Pb inhibits microglial migration by downregulation of SOCE and impairment of the function of STIM1.

Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1

Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca2+ sensor and activator of Orai1 channels. In resting cells with replete Ca2+ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca2+ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca2+ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.

Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1.

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it toward the plasma membrane to activate Orai and SOCE after store depletion.

The inactivation domain of STIM1 acts through intramolecular binding to the coiled-coil domain in the resting state.

Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that is controlled by the ER Ca2+ sensor STIM1. Abnormal activation of STIM1 directly influences Ca2+ influx, resulting in severe diseases such as Stormorken syndrome. The inactivation domain of STIM1 (IDstim) has been identified as being essential for Ca2+-dependent inactivation of STIM1 (CDI) after SOCE occurs. However, it is unknown whether IDstim is involved in keeping STIM1 inactive before CDI. Herein, we show that IDstim helps STIM1 keep inactive through intramolecular binding with the coiled-coil domain. Between IDstim and the coiled-coil domain, we found a short conserved linker whose extension or mutation leads to the constitutive activation of STIM1. We have demonstrated that IDstim needs the coiled-coil domain 1 (CC1) to inhibit the Ca2+ release-activated Ca2+ (CRAC) activation domain (CAD) activity and binds to a CC1-CAD fragment. Serial deletion of CC1 revealed that CC1α1 is a co-inhibitory domain of IDstim. CC1α1 deletion or leucine mutation, which abolishes the closed conformation, impaired the inhibitory effect and binding of IDstim. These results suggest that IDstim cooperates with CC1α1 to help STIM1 keep inactive under resting conditions.

STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.

Oligomerization of the Ca2+ sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca2+ stores, results in STIM1 coupling to the plasma membrane Ca2+ channel protein, Orai1, to activate Ca2+ influx in a process known as store-operated Ca2+ entry. We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal (N-terminal) or the cytoplasmic (C-terminal) ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain (CAD/SOAR, human sequence 342–448) is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1–448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449–462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.

A protease-independent function for SPPL3 in NFAT activation.

The signal peptide peptidase (SPP)-related intramembrane aspartyl proteases are a homologous group of polytopic membrane proteins, some of which function in innate or adaptive immunity by cleaving proteins involved in antigen presentation or intracellular signaling. Signal peptide peptidase-like 3 (SPPL3) is a poorly characterized endoplasmic reticulum (ER)-localized member of this family, with no validated cellular substrates. We report here the isolation of SPPL3 in a screen for activators of NFAT, a transcription factor that controls lymphocyte development and function. We find that SPPL3 is required downstream of T cell receptor engagement for maximal Ca(2+) influx and NFAT activation. Surprisingly, the proteolytic activity of SPPL3 is not required for its role in this pathway. SPPL3 enhances the signal-induced association of stromal interaction molecule 1 (STIM1) and Orai1 and is even required for the full activity of constitutively active STIM1 variants that bind Orai1 independently of ER Ca(2+) release. SPPL3 associates with STIM1 through at least two independent domains, the transmembrane region and the CRAC activation domain (CAD), and can promote the association of the STIM1 CAD with Orai1. Our results assign a function in lymphocyte signaling to SPPL3 and highlight the emerging importance of nonproteolytic functions for members of the intramembrane aspartyl protease family.

Testing a Diffusion Trap Model for Store-Operated Calcium Entry by Single Particle Tracking

Depletion of calcium from the endoplasmic reticulum (ER) triggers autonomous assembly of STIM1-Orai1 complexes at ER-plasma membrane (PM) junctions, leading to store-operated calcium entry (SOCE). One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions by binding to the PM via its C-terminal polybasic domain. STIM1 then traps Orai1 in the PM through binding of STIM1's CRAC activation domain. We tested this diffusion trap model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian with a mean diffusion coefficient (D) of 0.12 µm2/s, while Orai1 is slightly subdiffusive (mean D=0.09 µm2/s). After store depletion both proteins slow to the same speed (mean D≈0.03 µm2/s), consistent with complex formation, and are confined to an area similar in size to ER-PM junctions. At high STIM1:Orai1 expression ratios, the probability that Orai1 escapes the diffusion trap is >1%, but the escape probability is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM1 and Orai1 are trapped by their physical connections across the junctional gap. However, we also find that STIM-Orai interactions are surprisingly dynamic in that readily reversible binding reactions generate free STIM1 and Orai1 which can escape and exchange with extrajunctional pools. These data suggest that STIM1 and Orai1 interact with low affinity, creating the potential for SOCE modulation even when ER calcium stores are mostly depleted, and promoting the reversal of SOCE upon store refilling.

Single-Molecule Studies of the ER Calcium Sensor STIM1

The ER membrane (EM) protein STIM1 is the ER Ca2+ sensor that initiates store-operated Ca2+ entry through Orai channels in lymphocytes and many other cells. Communication of ER Ca2+ store depletion to Orai channels in the plasma membrane involves a transition of STIM1 dimers from an inactive to an active state, but the conformational changes underlying this transition remain poorly characterized. We have developed an in-vitro assay to study the conformation and dynamics of STIM1 at the single-molecule level. Purified STIM1 cytosolic fragments were labeled at single sites with Alexa555 and Alexa647 by site-directed mutagenesis and cysteine-maleimide chemistry. These served as donor and acceptor dyes for high-resolution intradimer distance measurements using single-molecule FRET. Labeled STIM1 fragments were encapsulated in 100nm diameter, surface-immobilized liposomes and imaged with a prism-based TIRF microscope.We identified three broad regions within the STIM1 fragments, defined by their characteristic FRET signatures. Within the ER-proximal N-terminal region containing the predicted coiled-coil 1 domain (aa274-339), we observed several transiently stable conformational states, ranging from closely apposed (FRET ∼0.8) to widely separated (FRET ∼0). The different FRET states occurred with similar probability, and transitions between them were frequent. In the region 363-449, encompassing the CRAC activation domain (CAD), stable intermediate to high FRET values (0.6-0.9) were dominant, indicating close apposition of paired sites in the dimer. Interestingly, low-FRET states (0.1-0.4) were also observed at several sites, suggesting the existence of an open conformation with relatively low probability. Finally, C-terminal to the CAD (aa512-660) we found stable, broad low-FRET signals, consistent with the predicted unstructured nature of this region. FRET ratios progressively declined from ∼0.2 to 0 towards the C-terminal end, indicating increasing separation. Further development of this approach will enable direct, high-resolution probing of STIM1 activation under controlled conditions.

Single-Molecule FRET Studies of the ER Calcium Sensor STIM1

The ER membrane (EM) protein STIM1 is the ER Ca2+ sensor that initiates store-operated Ca2+ entry through Orai channels in lymphocytes and many other cells. Communication of ER Ca2+ store depletion to Orai channels in the plasma membrane involves a transition of STIM1 dimers from an inactive to an active state, but the conformational changes underlying this transition remain poorly characterized. We have developed an in-vitro assay to study the conformation and dynamics of STIM1 at the single-molecule level. Purified STIM1 cytosolic fragments were labeled at single sites with Alexa555 and Alexa647 by site-directed mutagenesis and cysteine-maleimide chemistry. These served as donor and acceptor dyes for high-resolution intradimer distance measurements using single-molecule FRET. Labeled STIM1 fragments were encapsulated in 100nm diameter, surface-immobilized liposomes and imaged with a prism-based TIRF microscope. We identified three broad regions within the STIM1 fragments, defined by their characteristic FRET signatures. Within the ER-proximal N-terminal region containing the predicted coiled-coil 1 domain (aa274-339), we observed several transiently stable conformational states, ranging from closely apposed (FRET ∼0.8) to widely separated (FRET ∼0). The different FRET states occurred with similar probability, and transitions between them were frequent. In the region 363-449, encompassing the CRAC activation domain (CAD), stable intermediate to high FRET values (0.6-0.9) were dominant, indicating close apposition of paired sites in the dimer. Interestingly, low-FRET states (0.1-0.4) were also observed at several sites, suggesting the existence of an open conformation with relatively low probability. Finally, C-terminal to the CAD (aa512-660) we found stable, broad low-FRET signals, consistent with the predicted unstructured nature of this region. FRET ratios progressively declined from ∼0.2 to 0 towards the C-terminal end, indicating increasing separation. Further development of this approach will enable direct, high-resolution probing of STIM1 activation under controlled conditions.

Stim1 Binds to Pairs of Orai1 Subunits to Open the Crac Channel

CRAC channels are activated by binding of the ER Ca2+-sensor STIM1 to the cytoplasmic C-terminus of the channel subunit Orai1. The crystal structure of Drosophila Orai describes a trimer of Orai dimers in which each C-terminus forms an antiparallel coiled-coil with its neighbor. This unexpected arrangement raises the question of whether pairs of C-termini cooperate to bind STIM1 and thereby function as a unit, or whether single C-termini act independently to bind STIM1 and activate Orai1.We assayed binding by E-FRET between CFP-labeled Orai1 tandem dimers and YFP-labeled STIM1 CRAC activation domain (aa#342-448) expressed in HEK cells. FRET indicated strong binding to normal (WT-WT) dimers and no binding to L273D-L273D dimers, as expected (the C-terminal L273D mutation prevents STIM1 binding). Dimers with one C-terminus deleted (WT-CT) had weak FRET, indicating that STIM1 binds weakly to the 3 monomeric C-termini in the assembled channel. However, WT-L273D heterodimers produced significantly higher FRET, showing that the “non-binding” L273D C-terminus contributes to STIM1 binding when paired with a WT C-terminus. These results suggest that STIM1 binds to pairs of Orai1 C-termini in the native channel. To quantify the effects of L273D on Orai1 activation, we generated hexameric concatemers with L273D mutations in subunits 1, 1+3, or 1+3+5, producing channels with one, two, or three heterodimers, respectively. When co-expressed with saturating levels of STIM1, WT concatemers generated normal CRAC-like currents. L273D reduced the current magnitude in a highly nonlinear manner; each heterodimer reduced activity by ∼2/3, while the three-heterodimer channel produced no detectable current. Thus, although STIM1 can bind WT-L273D heterodimers, the binding energy provided does not open the channel. Future experiments will test whether L273 acts simply to bind STIM1 or whether it also contributes to coupling binding to channel opening.

The C‐ and N‐terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Distinct Functional Roles of the N- and C-Terminal STIM1 Binding Sites in Orai1 for Trapping and Gating of CRAC Channels

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of the ER Ca2+ sensor, STIM1, and the CRAC channel protein, Orai1, to overlapping sites in the ER- and plasma-membranes. Redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, a critical step that not only controls CRAC channel gating, but also regulates its pore properties including calcium selectivity. Previous findings indicate that Orai1 harbors two STIM1 binding sites, located in the intracellular N- and C-termini. Here, we compared the functional contributions of these sites for puncta formation, gating, and modulation of CRAC channel ion selectivity. Consistent with past studies, C-terminal mutations that impaired STIM1-Orai1 association and puncta formation also abrogated channel gating. However, gating was fully restored in some of these mutants by directly tethering a STIM1 fragment containing the minimal CRAC activation domain (CAD) in duplicate to Orai1 (Orai1-SS channels). By contrast, mutants lacking the N-terminal CAD binding site displayed no channel activity despite retaining significant STIM1-Orai1 binding, and this defect could not be restored in Orai1-SS channels. Analysis of ion selectivity indicated that the N-terminal site is essential for conferring high calcium selectivity to Orai1 while the C-terminal site is dispensable if CAD is directly linked to Orai1. Thus, the structural requirements for gating and modulation of ion selectivity are similar but substantively different from those for Orai1 puncta formation. Collectively, these results are broadly consistent with a two-step model for Orai channel activation, in which the C-terminal binding site tethers and retains CAD to Orai1, thereby allowing functionally critical interactions at the Orai1 N-terminus to gate the channel and modulate its pore properties.

Orai-Stim Mediated Ca2+ Release from Secretory Granules Revealed by a Novel Ca2+ and pH Probe

Secretory granules (SGs) bud from the trans-Golgi network and during subsequent maturation, acquire an acidic intraluminal pH, electron-dense cores, and matrices that bind Ca2+ and other ions. Total Ca2+ in SGs is remarkably high, 30-40 mM. The free Ca2+ is significantly lower, with estimates ranging from 10-80 μM. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty in measuring SG calcium directly in living cells. We adapted the FRET-based D1-ER probe to develop a novel probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca2+ of 69 ± 15 μM and pH 5.8. Application of ATP resulted in a slow monotonic decrease in SG Ca2+ temporally correlated with the occurrence of store-operated calcium-entry (SOCE). Further investigation revealed a novel receptor-mediated mechanism of calcium release from SGs that involves store-operated channels. SG Ca2+ release is completely antagonized by a SOCE antagonist, by switching to Ca2+-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca2+ by ∼75% and completely abolished the ATP-mediated release of Ca2+ from SG's. Overexpression of a dominant negative CAD construct (CAD-A376K) induced no significant changes in SG Ca2+. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels, and during SG Ca2+ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SGs membranes as a potential new mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the endoplasmic reticulum and exocytosis. (Grants NS08174/GM83913/F32 DC068982/K01 MH086119/NSFChina/985DEChina).

Changes in STIM1 Interactions during Activation of Store-Operated Ca2+ Entry Monitored by FRET

Oligomerization of STIM1 and co-clustering with the Ca2+ channel protein, Orai1, at ER-plasma membrane (PM) junctions activates Ca2+ influx known as store-operated Ca2+ entry (SOCE) that is associated with visible puncta formed in the plane of the PM. Current information suggests that STIM1 in the resting state is minimally dimeric due to interactions between cytoplasmic CRAC activation domain (CAD) segments. We previously identified a basic sequence inside CAD responsible for activation of Orai1, and an acidic inhibitory region flanking CAD that controls a conformation change during activation. In recent studies we developed a fluorimetry-based fluorescence resonance energy transfer (FRET) method to monitor STIM1 oligomerization, both at the luminal (N-terminal) side, as well as at the cytoplasmic (C-terminal) side of STIM1, and we probed these interactions with mutations in STIM1 that are tagged with fluorescent protein reporterss. We found significant differences in stimulated FRET during Ca2+ store depletion depending on where the fluorescent proteins were placed: N-terminally tagged STIM1s exhibits stronger stimulated FRET than C-terminally tagged derivatives. Co-expressing untagged Orai1 largely eliminates stimulated FRET between C-terminally-tagged STIM1, but does not reduce stimulated FRET at the luminal side. Functional coupling between AcGFP-Orai1 and STIM-mApple can be monitored either by FRET or by Ca2+ influx using the Ca2+ sensor GCaMP3. Sequential truncations of STIM1 from the C-terminus support a role for CAD as a oligomer-promoting part of STIM1. Our FRET results showing nanometer-scale re-arrangements of STIM1 oligomers during activation provide a higher resolution picture that is distinctive from micrometer scale clustering that is visualized as puncta formation.

The Third Transmembrane Segment of Orai1 Protein Modulates Ca2+ Release-activated Ca2+ (CRAC) Channel Gating and Permeation Properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Role of the STIM1 C-terminal Domain in STIM1 Clustering

Store-operated Ca(2+) entry (SOCE) represents a ubiquitous Ca(2+) influx pathway activated by the filling state of intracellular Ca(2+) stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca(2+) sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca(2+) stores are full, these domains are inactive to prevent constitutive Ca(2+) entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca(2+) stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.

Stoichiometric Requirements for Trapping and Activation of CRAC Channels by STIM1 At ER-Plasma Membrane Junctions

Store-operated Ca2+ entry is regulated by physical interactions of the ER Ca2+ sensor STIM1 and the CRAC channel protein Orai1. Recent studies support a diffusion-trap mechanism in which Ca2+ depletion from the ER causes STIM1 to accumulate at ER-plasma membrane (ER-PM) junctions, where its CRAC activation domain (CAD) binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. The ability of the CAD to cluster CRAC channels suggests that each channel contains multiple STIM1 binding sites, but the number of sites that must be occupied to trap and activate CRAC channels is unknown. By competing for a limited amount of STIM1, increasing levels of Orai1 are expected to reduce the number of STIM1s bound per channel until the minimum stoichiometry sufficient for channel trapping is reached. In HEK293 cells expressing a fixed amount of mCherry-STIM1 and increasing levels of GFP-Orai1, the junctional STIM1:Orai1 ratio reached a minimum of ∼0.3; thus, binding of 1-2 STIM1 appears sufficient to immobilize tetrameric CRAC channels at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current (ICRAC) was a highly nonlinear bell-shaped function of Orai1 expression, and the minimum STIM1:Orai1 stoichiometry for channel trapping failed to evoke significant activation. A simple cooperative gating model fitted to the data suggests that while STIM1 binding to a single site is sufficient to trap CRAC channels, four sites must be occupied to cause significant activation. This highly nonlinear behavior supports conclusions based on current fluctuation analysis (Prakriya and Lewis, J. Gen. Physiol. 128:373-86, 2006) that the slow development of whole-cell ICRAC after store depletion reflects the stepwise recruitment of individual channels from a silent to a high open-probability state as they enter ER-PM junctional sites.

Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1

Oligomerization of the ER Ca(2+) sensor STIM1 is an essential step in store-operated Ca(2+) entry. The lumenal EF-hand and SAM domains of STIM1 are believed to initiate oligomerization after Ca(2+) store depletion, but the contributions of STIM1 cytosolic domains (coiled-coil 1, CC1; coiled-coil 2, CC2; CRAC activation domain, CAD) to this process are not well understood. By applying coimmunoprecipitation and fluorescence photobleaching and energy transfer techniques to truncated and mutant STIM1 proteins, we find that STIM1 cytosolic domains play distinct roles in forming both "resting" oligomers in cells with replete Ca(2+) stores and higher-order oligomers in store-depleted cells. CC1 supports the formation of resting STIM1 oligomers and appears to interact with cytosolic components to slow STIM1 diffusion. On store depletion, STIM1 lacking all cytosolic domains (STIM1-DeltaC) oligomerizes through EF-SAM interactions alone, but these oligomers are unstable. Addition of CC1 + CAD, but not CC1 alone, enables the formation of stable store-dependent oligomers. Within the CAD, both CC2 and C-terminal residues contribute to oligomer formation. Our results reveal a new function for the CAD: in addition to binding and activating Orai1, it is directly involved in STIM1 oligomerization, the initial event triggering store-operated Ca(2+) entry.

Bombyx mori Homologs of STIM1 and Orai1 Are Essential Components of the Signal Transduction Cascade That Regulates Sex Pheromone Production

Sex pheromone production in the pheromone gland (PG) of the silkmoth, Bombyx mori, is mediated by store-operated channels (SOCs) acting downstream of pheromone biosynthesis activating neuropeptide (PBAN) binding. Although recent studies have implicated STIM1 and Orai1 as essential components of SOCs, little is known about the molecular nature of the SOCs involved in sex pheromone production. In this study we cloned silkmoth homologs of STIM1 and Orai1 and sought to determine whether they comprise the PG SOC pathway. BmSTIM1 is expressed in multiple tissues and, in the PG, is encoded by two transcripts of differing size. BmOrai1A and BmOrai1B, which are identical except for a 37-residue N-terminal truncation in BmOrai1B, arise from alternative splicing of the bmorai1 locus and are expressed as independent transcripts in various tissues. In the PG, only BmOrai1B is actively transcribed. Fluorescent chimeras demonstrated that BmSTIM1 expression is restricted to the endoplasmic reticulum, whereas both BmOrai1A and BmOrai1B localize to the cell surface. In Ca(2+)-free medium, thapsigargin-mediated depletion of endoplasmic reticulum Ca(2+) stores resulted in redistribution of BmSTIM1 to the plasma membrane, but only when the BmOrai1 homologs were also overexpressed. Translocation was dependent on the BmSTIM1 C terminus "CRAC activation domain." Ala mutation of Lys(380), Lys(383), Lys(384), Arg(382), and Arg(385) suggests that translocation involves electrostatic interactions. Translocation was also seen following PBAN stimulation in cells co-expressing BmSTIM1, BmOrai1B, and the PBAN receptor. In vivo RNA interference-mediated knockdown of BmSTIM1 and BmOrai1 significantly reduced sex pheromone production without affecting cell viability.

STIM1 and calmodulin interact with Orai1 to induce Ca 2+ -dependent inactivation of CRAC channels

Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.