The Gill lab is focused on understanding the cellular and molecular basis of calcium signal transduction. Our work centers on the process known as “store-operated calcium entry”, or SOCE. This process is key for regulating a diverse set of signaling pathways and is essential in virtually all eukaryotes (1-3). Transient depletion of ER Ca2+ stores is sensed by the Ca2+ binding EF-hands of the single-pass transmembrane protein STIM1 (4). Upon sensing these changes, STIM1 undergoes an intricate unfolding process that allows the lysine-rich C-terminus to elongate and interact with PM phosphatidylinositol 4,5-bisphosphate residues (1,2,4-7). Interactions with these phospholipids leads to STIM1 trapping within ER-PM junctions, and promotes subsequent tethering and activation of Orai1 channels that passively migrate through the PM (5). This dynamic coupling process is critical to the formation of discrete Ca2+ microdomains within ER-PM junctions (1,2,4). Ca2+ signals generated through active Orai1 channels control a diverse array of cellular processes such as cell growth, cell motility, transcription, secretion, and the maintenance of intracellular Ca2+ homeostasis. Ca2+ actively pumped back into the ER via the sarco/endoplasmic reticulum ATPase (SERCA) pump allows stores to be replenished. In recent years, much progress has been made towards deciphering the key details behind the STIM1-Orai1 interaction, specifically regarding Orai1 activation. What is unclear, however, is precisely how STIM1 coupling to Orai1 leads to channel gating and subsequent Ca2+ entry (2,7,8). We will use this chapter to describe some recent advances in our laboratory toward better understanding STIM1-Orai1 coupling and channel gating.
Figure 1: The STIM1-Orai1 interaction mechanism. The inactive form (left) of the STIM1 dimer remains folded in on itself at resting Ca2+ concentrations in the endoplasmic reticulum (ER). High concentrations of Ca2+ cause inactivation of the STIM proteins through binding to the EF-hand domain of STIM. The closed form of Orai1 (left) can freely diffuse throughout the plasma membrane (PM). Upon ER Ca2+ store depletion however, STIM proteins unfold and extend to associate with the PM and bind Orai1 directly (right) to cause Ca2+ entry into ER-PM junctions. The cytosolic facing segments of STIM1 include the lysine-rich (K-rich) C-terminal domain that binds to phospholipids in the PM, and the flexible C-terminal domains (flex C-term) that aid in shielding the STIM-Orai activating region (SOAR) during store replete conditions. The luminal/intramembranous segments include the transmembrane region (TM) that transmits conformational changes induced by the sterile-α-motif (SAM) and Ca2+-binding EF-hand domain.
Two major components of the store operated Ca2+ entry pathway are the membrane-spanning ER STIM proteins (STIM1 and STIM2) and the family of Orai Ca2+ entry channels (4). The STIM proteins naturally exist as dimers at resting ER Ca2+ concentrations. In their inactive forms, the C-terminal cytosolic portion of STIM proteins are folded in upon themselves, occluding the Orai1 binding domain and preventing trapping within ER-PM junctions Fig. 1 (9-11). Decreases in ER luminal Ca2+ concentrations are recognized by the two N-terminal Ca2+ binding EF hand domains that reside in the ER lumen. Transient ER luminal Ca2+ depletion results in a STIM protein conformational change, whereby the luminal N-termini of the dimeric proteins become closer associated. This conformational change induces the C-terminal cytosolic portion of the dimeric STIM protein to unfold and extend into the cytosol and be able to associate with the PM in ER-PM junctions (12). The unfolded C-termini of dimeric STIM are highly conserved between the two STIM isoforms in a region termed the STIM-Orai activating region (SOAR; 344-442) (13). Slightly extended versions of this region are known as the channel-activating domain (CAD; 342-448) (5), and Orai1-activating small fragment (OASF; 233-450) (9). SOAR is the smallest functional unit of STIM, and remarkably can be expressed alone as a soluble cytoplasmic protein to fully activate the Orai1 channel (13). Structures derived from X-ray crystallography of purified SOAR fragments reveal that it exists as a highly structured dimer (14). Each monomer is mainly helical in nature and is composed of four core α-helices, or eight in total between the two subunits in a dimer (Fig. 2).
Figure 2. The crystal structure of the dimeric SOAR domain. The dimeric SOAR domain comprises four α-helical regions (α1, α2, α3, α4). The purported strong activation site resides between helixes α1 and α3, and includes the Phe-394 residue. The α4 helical region is critical for maintaining the structure of SOAR dimers, as mutations to the α4 resident R429 residue prevents SOCE and initiates aberrant STIM1 unfolding in human patients. Our lab has substituted residue Phe-394 to measure its importance to the STIM1-Orai1 interaction (lower panel). Changing the residue to leucine (equivalent residue in STIM2), alanine, or histidine results in abrogation of the interaction with Orai1 and prevention of SOCE.
This core helical structure is purportedly conserved within the C-terminus of full-length extended STIM1, and empirical evidence suggests that it is critical for dimer formation and maintaining the dimeric interaction between monomers of STIM1 (9-11,14). The exposed SOAR domain is free to bind Orai1 in the PM. Binding to Orai1 channels causes transient trapping and activation of the channels within ER-PM junctions (11,14,15). Ca2+ brought in through Orai1 trapped in ER-PM junctions potentiates calcineurin-induced NFAT-dephosphorylation, which regulates transcription of a large number of genes in many different cell types (16,17). The structure of the Drosophila Orai channel (dOrai) is also now known in some detail (Fig. 3).
Figure 3. Structure of the Drosophila Orai (dOrai) channel. The dOrai channel has close homology with the mammalian Orai1 channel and likely very similar structure. The crystal structure does not include the N-terminal cytosolic sequence (equivalent to the first 60 residues in human Orai1) which is largely redundant to channel function and activation by STIM1. The Orai channel largely has 6-fold symmetry, with six subunits each with identical sequences. Each subunit has for transmembrane helices (TM1 through TM4). The TM1 helix forms the channel pore (yellow) and the outer channel selectivity filter glutamate is shown (E178 in dOrai1, or E106 in human Orai1). The other TM helices are packed around the central pore helix, with TM4 (red) toward the outside. The C-terminal helical extension of TM4 (TM4-ext) extends at the periphery of the channel and is joined to the TM4 through a “nexus” region (light blue). This nexus includes a hinge that is configured in two different ways: three TM4 helices are in a “straight” hinge configuration (A1, A2, and A3), and three are in a bent config-uration (B1, B2, and B3) as shown in the diagram. Thus, the Orai hexamer includes three dimers, each dimer comprising one “A” and one “B” form of the channel. As are result, the two adjacent TM4-ext A and B helices in each dimer are closely aligned in an anti-parallel manner and linked through hydrophobic reactions between the L273 and L276 residues These residues and the outer Orai TM4-ext configuration which constitutes the STIM binding site are described further in Fig. 5.
X-ray crystallographic structures of the purified PM localized dOrai channel reveal it to be a homo-hexamer (18,19), with each monomeric Orai1 subunit being composed of four transmembrane-spanning domains (TM1-TM4) (18). The central pore of the hexamer is lined by the N-terminal TM1 (18). TM2 and TM3 are tightly packed around the pore, while the C-terminal TM4 is less tightly packed and in fact extends partially into the cytosol and is the strong binding site for the STIM proteins (5,18). Unfortunately there are not as yet any solved structures of STIM bound to Orai1.
The precise features of the STIM-Orai coupling interaction are still unclear and currently a major focus of many labs (1,2,7,8,20). Nevertheless, a number of studies using a combination of approaches including analysis of fluorescently labeled Orai and STIM fragments, basic electrophysiology and biochemistry, have lead to some significant advances in understanding the stoichiometry of STIM1-Orai1 coupling. Interestingly, these experiments have lead to the conclusion that the stoichiometry of interaction can vary, with maximum activation occurring at a STIM1:Orai1 ratio of 2:1 (21-23). However, recent structural studies utilizing partial SOAR and C-terminal STIM1-binding Orai1 TM4 fragments suggest that binding occurs in a bimolecular fashion whereby a STIM1 dimer will bind to two adjacent Orai1 subunits within the same Orai1 hexamer (24-26). This model would indicate a stoichiometry of 1:1, yet it cannot explain the variable stoichiometry seen in previous experiments. Follow-up studies in our laboratory are described in detail below, and lend support to the unimolecular model, indicating a 2:1 STIM1:Orai1 stoichiometry.
To better understand the STIM1-Orai1 coupling interaction, we undertook a comparative analysis of the SOAR regions of STIM1 and STIM2 because of the natural differences in affinity for Orai1 between STIM1 and STIM2, with STIM1 being the higher affinity isoform. We observed almost complete homology between the two isoforms except at position Phe-394 in STIM1 (Fig. 2) and the equivalent position Leu-485 in STIM2. Experiments in our lab indicate that this residue plays an important role in the reduced affinity that STIM2 has for Orai1 (27). Mutation of Phe-394 in STIM1 to alanine resulted in a strong reduction of channel activity and strong decreases in the interaction. Surprisingly, the relatively conservative mutation to histidine (Fig. 2, lower) almost completely abolished STIM1 binding to Orai1 and completely abolished Ca2+ entry (27), yet had no effect on the resting state of STIM1 or its ability to be activated by transient ER Ca2+ depletion.
Figure 4. An alternative unimolecular STIM1-Orai1 coupling model. Previous studies have suggested a bimolecular STIM1-Orai1 model for coupling (A) model, in which the two active sites of the SOAR dimer unit must both interact with two adjacent Orai1 subunits of the hexamer in order to open the channel. Only two Orai1 subunits from the Orai1 channel hexamer are shown. The SOAR dimer is depicted as the concatemer with two Phe-394 wildtype residues with YFP attached. In our unimolecular STIM1-Orai1 model for coupling (B), just one of the SOAR subunits in the dimer is necessary to bind to a single subunit of the Orai1 hexamer to gate the channel. The diagram shows a concatenated SOAR dimer with one wildtype and one mutated F394H SOAR subunit. (C) Depiction of the theoretical interaction of SOAR dimers with the hexameric Orai1 channel. A unimolecular model for coupling envisages that a wildtype SOAR homodimer and the heterodimer of SOAR with one SOAR-F394H mutant unit (blue), would both be able to interact with and completely activate Orai1. (D) Possible clustering of hexameric Orai1 channels mediated by cross-linking by wildtype SOAR dimers.
Our lab has developed novel SOAR probes that included the mutation at residue Phe-394. Using these probes, we identified this residue as being a vital component of the Orai1-binding site (28). By genetically encoding a flexible linker between each SOAR subunit, we were able to make efficiently expressed SOAR dimers and precisely control the stoichiometry of mutant to wild-type SOAR within a construct (Fig. 4A). We constructed a series of concatenated dimers containing either one or both of the Phe-394 residues mutated to histidine in each SOAR peptide, and used these to test the foundations of the bimolecular interaction model. This model would predict that heterodimers containing a subunit with the F394H mutation would be nonfunctional, however to our surprise we observed similar activity between wild-type homodimers and mutant heterodimers (28). This result suggests that Orai1 channel activity is dependent on only one functionally active site within a SOAR dimer, which is not predicted by the bimolecular model.
While SOAR is always a dimer in both STIM1 and concatenated peptides, only one functional subunit appears required for interaction and full activation of Orai1 channels. Nevertheless, dimerization of STIM1 is key to its function in activating Orai1. Mutations that alter dimerization have a major impact on the ability of STIM1 to couple with and activate Orai1 (14). An interesting mutation in the SOAR segment (Fig. 2), R429C, reveals that this residue is crucial for maintaining the structural integrity of SOAR even though mutating it does not cause dimer dissociation. Patients expressing STIM1 containing the naturally occurring but rare R429C mutation experience a complete loss in STIM1 function (26). This residue was initially identified as being structurally important in the crystal structure of SOAR, as it resides within the α4 helix within an area involved in maintaining intermolecular protein interactions (Fig. 2). The occurrence of the mutant in patients highlighted its importance in promoting the stability of the SOAR dimer region, which has been shown to be critical for SOAR binding to Orai1 channels (14,24,26). Subsequent experiments from constructs expressing STIM1 (R429C) demonstrated that mutant had a dual effect on blocking STIM1-Orai1 coupling and also caused constitutive localization of STIM1 into ER-PM junctions (26). This suggests that the Arg-429 residue is critical to maintaining the resting folded state of STIM1, and prevents STIM1 from unfolding without the appropriate signal from depleted ER Ca2+ stores.
The results from our studies using the F394H mutation in SOAR indicate that its mode of function is entirely different from R429C (28). While the R429C mutant is constitutively aggregated into ER-PM junctions, STIM1 F394H protein maintains its folded state until stores are depleted. Only after store depletion does the F394H mutant move into junctions, and there within junctions it is functionally deficient in coupling to Orai1 channels which is necessary for channel gating. Based on the crystal structure, the Phe-394 residue is located towards the apex of each SOAR monomer (Fig. 2), whereas the Arg-429 residue is located deep within the structure and appears to be solely involved in intramolecular interaction between each peptide helices (Fig. 2).
Experiments utilizing novel SOAR probes suggest a coupling mechanism entirely different than what was previously proposed. The previous bimolecular model dictates that SOAR dimers bind across two adjacent Orai1 subunits within the same hexamer to activate the channel (Fig. 4A). This was supported by NMR studies that used shorter fragments of the STIM-Orai1 binding domain and a portion of the C-terminus STIM1 binding site of Orai1 (24,25). These studies using short SOAR-derived peptides, however, selected regions of SOAR that were unable to activate Orai1 when coexpressed, and did not include the high affinity region surrounding the Phe-394 residue that was shown to be critical for STIM1-Orai1 coupling in our experiments (24,25). Results from our lab that utilized SOAR concatemeric dimers containing F394H mutations to one, or both subunits, suggest that a “unimolecular” interaction between SOAR/STIM and Orai1 is necessary and sufficient for channel coupling and gating (Fig. 4B). These results were the first demonstration that a single binding site within a SOAR dimer is sufficient for binding to adjacent Orai1 hexamers and not within the same hexamer as predicted by the “bimolecular model”.
To examine the actual binding in more detail, and to clarify the unimolecular interaction model, we probed the ability of each SOAR dimer to independently interact with the C-terminal STIM1-binding site on TM4 of Orai1 channels. The cytosolic C-terminal peptide of Orai1 has long been established as the strong binding site for STIM1/SOAR (5,29). We designed a novel construct encompassing 35 amino acids from the C-terminal helix (Orai1CT; residues 267-301) labeled with CFP. Within the N-terminus of CFP we encoded a PM-directed single transmembrane-spanning helical peptide (PMP), and expressed these constructs in HEK cells along with our assortment of YFP-labeled SOAR concatemer probes (constructs comprising either two wild-type binding domains, one mutant F394H in either domain, or a double mutant F394H) (Fig. 4B,C) (28). We used quantitative E-FRET to analyze the proximity between PMP-CFP-Orai1CT and the different YFP-SOAR concatemeric constructs. To our surprise, we found that homodimers of the wild-type SOAR binding domain bound roughly two PMP-CFP-Orai1CT molecules, whereas the heterodimers were only able to bind to one. These results definitively show that SOAR dimers can bind across at least two separate STIM-binding sites located on the C-termini of Orai1 subunits.
Our results from the quantitative E-FRET experiments lead us to question the need for a second site for STIM1 to bind to on Orai1 channels, and strongly corroborated earlier functional studies undertaken in our lab. Interestingly, if a SOAR dimer within full-length STIM1 or soluble SOAR concatemers can bind across two Orai1 C-termini, then they may play a role in bridging separate Orai1 channels within ER-PM junctions. This bridge between Orai1 channels may also be a way of cooperatively regulating Orai1 channel activity, both through activation and inactivation, although there is no data to support this hypothesis yet. We hypothesize, however, that the SOAR domain within STIM1 can cross-link adjacent Orai1 channels to promote channel clustering (Fig. 4D). Along with our E-FRET data supporting the notion that SOAR dimers can bind across two Orai1 C-termini, other labs have previously shown that the longer CAD fragment of STIM1 could cluster Orai1 channels as indicated by electron microscopy (5). Another study utilized freeze-fracture electron microscopy to quantitatively measure the lateral distances between Orai1 channels and STIM1 after store depletion. The authors found that the mean space between Orai1 channels in ER-PM junctions was very similar to the mean distance (13-18nm) between STIM1 in the ER, implying that there may be a mechanism to promote organization of Orai1 and STIM in these junctions (30). Based on our results we would conclude that it is the act of STIM1 binding to Orai1 that can promote organization into clusters within the ER-PM junction (16,17).
As mentioned earlier, previous studies have noted the variable stoichiometry between STIM1 and Orai1 (21-23). The bimolecular coupling model has difficulty explaining variable stoichiometry between STIM1 and Orai1 because it mandates that the ratio of STIM1:Orai1 be 1:1, or 6 molecules of STIM1 per hexameric Orai1 channel. On the other hand, the unimolecular coupling can easily explain these observations. Using a hypothetical lattice between Orai1 and STIM1 dimers (Fig. 4D), we can easily predict that the stoichiometry varies as the size of a channel cluster gets bigger. Based on the size of the cluster, we would expect to see a variable STIM1-Orai1 stoichiometry ranging from 1:1 to 2:1. The stoichiometry would approach 1:1 the larger the cluster grows. The lattice model has interesting implications on the regulation of activation and deactivation kinetics of channels, and also on regulating the spatial disruption of channels to restrict Ca2+ entry signals to the ER-PM junctions. This may be a mechanism to discriminate complex signaling pathways as Ca2+ is a highly potent and cross-reactive second messenger.
Besides studying the interactions between STIM1 and Orai1, our lab has also focused on identifying how Orai1 channels transmit C-terminal binding of STIM1 into an open channel. STIM1 specifically binds to the C-terminal helical cytosolic extension of Orai1 TM4 (TM4-ext) (31). Using a mutational screen we identified a key region linking TM4 to TM3 and termed it the “nexus”. This nexus region is only five-amino acids long (residues 261-265 in hOrai1; LVSHK), and can transmit the allosteric binding signal from STIM1 to the channel core to initiate gating (Fig. 5). We found that mutating this nexus region from LVSHK to ANSGA caused the channel to be constitutively active and display all of the properties of a channel opened naturally by STIM1. We believe that this mutation exactly replicates the conformational change of TM4-ext normally caused by coupling with STIM1. The nexus is a discrete sequence and is composed of two fundamental regions. The first is the “hinge” domain (Ser-263, His-264, Lys-265) (18,32,33), and the second, the “hinge plate” (Leu-261, Val-262). Specifically, the residues of the hinge plate appear to be the hydrophobic attachment to TM3, and are the key transducers of the STIM1 binding signal. Previous groups have studied the hinge region by substituting with proline or cross-linking with cysteine through a redox reaction. These two alterations will lock the hinge and prevent the binding of STIM1 and Ca2+ entry through Orai1 (32,33). However, no group has studied the “hinge plate” region, which based on the crystal structure of Orai1 appears to be the main contact point between TM4 and TM3, specifically at residues Leu-261 from TM4 and Leu-174 from TM3. These two leucines form hydrophobic contacts that are critical for normal channel function. Substitution of either Leu-174 or Leu-261 with charged residues results in a block in STIM1-induced activation of Orai1 (31). Mutating these residues to cysteines and using a redox reagent to cross-link them enhanced channel activation (31). These results, coupled with the reports by other labs leads us to conclude that this nexus transduces the STIM1 coupling signal to trigger a conformational change from TM4 to TM3 and eventually TM2 and TM1. This leads to a rearrangement of the channel pore that allows Ca2+ to selectively enter. Experiments using Tb3+ luminescence in purified Orai1 protein confirms that cytosolic STIM1 binding leads to an immediate conformational alteration at the external face of the pore, specifically, movement near the selectivity filter Glu-106 and Val-102 (34).
Figure 5. Structure of Drosophila Orai1 dimers. Hydrophobic cross-interactions between Leu 273 and Leu-276 between antiparallel TM4 extensions (TM4-ext) of adjacent Orai1 dimers help hold the molecule together. The crystal reveals that each monomer is structurally homologous in each transmembrane (TM) region except for the TM4-ext between adjacent Orai1 monomers within a dimer. In the diagram above, TM4-A (light pink) is straight, and TM4-B (fuchsia) is bent. The TM4-ext of each Orai1 monomer is linked to the rest of the molecule at the hinge/nexus region (cyan). Our results from experiments utilizing the Orai1 nexus mutant (Orai1-ANSGA) suggest that STIM1 binding (1) causes dissociation of the TM4 extensions (2) and flexion on the hinge/nexus (3). This provides the force to displace the hinge plate residue (Leu-261) and subsequent displacement of the closely apposed Leu-174 residue on TM3 (4). Finally, displacement of Leu-174 would lead to conformational repositioning of core helixes TM3, TM2 and TM1 (5) and dilation of E106 selectivity filter In the Orai1 pore (6) to allow for selective Ca2+ entry.
We next used this model to ask whether the initial C-terminal interaction of STIM1 with Orai1 was sufficient to promote full channel activation, or does STIM1 require interaction with another region of the channel to cause activation. It has previously been shown that STIM1 can also interact weakly with the N-terminus (TM1) of Orai1 only after tethering to the Orai1 C-terminus to promote channel gating (5,8,29,32,34-40). Because the mutant ANSGA channel mimics the wild-type STIM1 activated channel, we used it to test whether the N-terminus of Orai1 is important for gating (31). The specific claim has been that residues within TM1 weakly interact with STIM1 to “pull open” the channel pore to allow Ca2+ entry (34). Specifically, Orai1 residues Leu-81, Ser-82 and Lys-85 have been implicated in this STIM1 interaction and are located within TM1 of Orai1. We introduced each of these mutations individually into the STIM1-idependent, constitutively opened Orai1-ANSGA channels, and also developed a construct containing all three mutations in the ANSGA Orai1 channel. We found that channel inhibition was similar in the ANSGA background to what was seen in wild-type STIM1 activated channels, suggesting that STIM1 interaction with these three TM1 residues is not necessary for channel gating but, rather, may be necessary for the integrity of the channel pore (31). It is more likely that mutation to these three residues disrupts channel function due to disruption of the pore architecture. These results support the notion that STIM1 interactions with the strong C-terminus binding site are necessary and sufficient for channel gating and Ca2+ entry. However we do not rule out the possibility that STIM1 interactions with the N-terminus are important for other mechanisms. Orai1-ANSGA channels do not experience Ca2+-dependent inhibition, which has been linked to the N-terminal interaction by STIM1 (31,41,42). Thus, it is likely that the N-terminal is important for mediating other aspects of channel activity but not the initial gating activity.
The discovery of the Orai1-ANSGA mutation has been extremely useful since it is a powerful tool for studying the role of STIM1 in promoting the Orai1 open channel conformation. It is also useful for determining the potential role that STIM1 plays in regulating other important aspects of channel activity such as Ca2+-dependent inhibition and the effect that localizing channels has on downstream signaling. This mutation is a superior alternative to other constitutively active Orai1 mutants as it exhibits channel characteristics almost identical to the wild-type STIM1 activated channel. Our lab was able to use the ANSGA construct as a tool to work out that STIM1 gating of Orai1 occurs primarily through its interactions with the C-terminal extension adjacent to the Orai1 nexus (Figs. 3 and 5). Based on the Orai1 crystal structure, we know that adjacent Orai1 monomers interact in a nonsymmetrical anti-parallel manner through their TM4-extension sequences. Specifically, the two adjacent TM4 extensions differ in their resting state conformations (Fig. 3). For one monomer, the hinge region is almost entirely straight (TM4-A) whereas in the other it is sharply bent (TM4-B) (Fig. 3). These extensions appear to be held together through hydrophobic interactions between Leu-273 and Leu-276, which are required for STIM1 binding (Fig. 5) (33). We propose that STIM1 binding to the Orai1 C-terminus acts by prying the TM4-extensions apart, perhaps through interactions with the Phe-394 residue in the SOAR active site that we described earlier. Displacement of these two M4-extensions may provide the force to flex the SHK-hinges, which initiates a force on the LV-hinge plate and displacement of the TM4 Leu-261 and TM3 Leu-174 residues. An analogy of this would be a classic lever system where STIM1 acts as the load, the C-terminus of Orai1 acts as the load arm, and the LVSHK hinge/hinge-plate region acts as the fulcrum. The concerted effort by these “lever” components generates a force that is transmitted through the LVSHK fulcrum through the tightly packed TM3/TM2/TM1 helices and allows for pore dilation and Ca2+ entry. Specifically, we believe that this force eventually leads to the reconfiguration of the extracellular-facing selectivity filter (Glu-106) to open the channel and occurs entirely through an allosteric mechanism that does not require any direct interaction by STIM1 to the N-terminus of Orai1. Further study will of course determine the authenticity of this model.
Overall, we propose a model for STIM1-induced Orai1 channel gating that exclusively involves the C-terminus of Orai1 (31). This model is different to previously suggested models involving both the C- and N-termini of Orai1 (5,8,29,32,34-40). We would suggest that development of the novel Orai1-ANSGA mutant provides us with a powerful tool to accurately study the role of STIM1 in Orai1 channel gating in ways that were previously impossible. We recognize the importance that the purported N-terminal STIM1 binding site (Leu-81, Ser-82 and Lys-85) may play on other channel properties, such as general maintenance of pore structure and/or Ca2+-dependent inhibition. However, results from our lab show that STIM1 interactions with this N-terminal region are unnecessary for channel gating. Our model provides a holistic view of the STIM1-Orai1 interaction mechanism (28,31). In summary, we propose that Orai1 channel activity is regulated by the unimolecular interaction between the monomeric active SOAR subunit in STIM1 dimers with the anti-parallel cytoplasmic C-terminal TM4-extensions of Orai1 channels. Clearly further study is required to better understand how allosteric interactions between STIM1 and the Orai1 C-terminus lead conformational changes at the Orai1 pore and transition into an active, Ca2+ selective Orai1 channel.
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