Fine tuning ICRAC: the interactions of STIM‐1 and Orai

Ca2+ release-activated Ca2+ channels (CRAC) are fundamental in the generation of sustained intracellular Ca2+ signals for a whole array of cellular functions. Ca2+ entry via CRAC was originally discovered in T-lymphocytes, but over the past 3 years investigations to elucidate the molecular components comprising CRAC have shown it to be a functional Ca2+ entry pathway in many cell types. The precise identification of these has been enigmatic, until recently. Activation of Ca2+ influx across the cellular plasma membrane, subsequent to depletion of Ca2+ from the endoplasmic (ER) or sarcoplasmic (SR) reticulum, is a phenomenon termed store-operated Ca2+ influx carried via store-operated Ca2+ channels or SOCs. Although the exact molecular identity of SOC remains elusive there is growing evidence for the involvement of both transient receptor channels (TRP) and CRAC. Both of these channels are prototypic SOC subtypes and, whilst the precise contribution of either of these subtypes remains controversial, a more pertinent question exists driving current research efforts; how does ER/SR store depletion communicate with and activate plasma membrane SOC? A recent burst of activity in the field has led to identification of Orai-1 as the fundamental CRAC pore-forming subunit in the plasma membrane (Yeromin et al. 2006). Stromal interaction molecule 1 (STIM-1), which at rest is diffusely present in the ER/SR membrane, was discovered to function as a sensor of ER Ca2+ concentration. Upon store depletion STIM-1 oligomerizes and is able to redistribute to discrete sites in the ER/SR membrane that are in close proximity to the plasma membrane (Luik et al. 2008). This study concluded that once STIM-1 oligomerized, the subsequent steps in activation of CRAC occur independently of ER/SR Ca2+ concentration. Currently, there is a lack of biochemical and structural studies on Orai, necessary to determine mechanisms of channel gating and ion permeation. Furthermore, the precise functional association between STIM-1 and Orai-1 remains unknown and is essential to fully elucidating the role of CRAC as SOC. Navarro-Borelly and colleagues published an interesting study in a recent issue of The Journal of Physiology using fluorescence resonance energy transfer (FRET) to define a direct redistribution and interaction of both STIM-1 and Orai-1 in response to store depletion of Ca2+ (Navarro-Borelly et al. 2008). In this elegant study they utilized a co-expression system of fluorescent protein tagged STIM-1 and Orai-1 in HEK cells. Their ultimate aim was to address the question of whether the accumulation of STIM-1 and Orai-1 at the ER–plasma membrane junctions brings them in sufficiently close proximity to enable a direct interaction and activation of CRAC. The authors first confirmed an association of STIM-1 and Orai-1 in response to ER store depletion by using either thapsigargin or cyclopiazonic acid, as inhibitors of the SR/ER Ca2+ pump (or Ca2+, Mg2+-ATPase), to deplete the ER Ca2+ store. They observed a redistribution of STIM-1 towards the cell periphery where it co-clustered with Orai-1. A striking increase in energy transfer detected by FRET was observed in parallel due to association between the STIM-1 and Orai-1 in discreet puncta (Navarro-Borelly et al. 2008). Using a similar time-lapse microscopy technique Navarro-Borelly and colleagues were then able to substantiate a multimeric stoichiometry of Orai-1. A robust constitutive FRET between co-expressed yellow fluorescent protein (YFP)- and cyan fluorescent protein (CFP)-labelled Orai-1 was observed in resting cells; this was independent of any potential FRET due to CFP-labelled CXCR4, used as an example of an unrelated plasma membrane channel subunit. Despite demonstrating a redistribution and association of STIM-1 and Orai-1 in response to store depletion, the molecular changes that constitute activation of a CRAC current (ICRAC) remained unresolved. Surprisingly, Navarro-Borelly et al. observed a reversible decrease in Orai–Orai FRET subsequent to store depletion which was abolished by a mutation in the EF-hand domain of STIM-1 (Navarro-Borelly et al. 2008). This decline in Orai–Orai FRET occurred simultaneously with the redistribution of Orai-1 into discrete puncta in the plasma membrane and was dependent upon the presence of STIM-1. Such interaction of STIM-1 and Orai in response to store depletion can therefore be directly associated with a rearrangement of the multimeric form of Orai-1. To further confirm that the decline in Orai–Orai FRET occurs in response to a physical rearrangement within the CRAC channel, two experiments were completed. Firstly, the position of the fluorescent tag was assessed; despite having a higher basal FRET, a CFP-tagged Orai-1 N-terminus resulted in a significantly smaller decline in FRET than the tag being positioned on the C-terminus. In addition, using elevated extracellular Ca2+ concentration to cause a Ca2+-dependent potentiation (CDP) of ICRAC decreased Orai–Orai FRET. Together these observations suggest a conformational rearrangement of the Orai C-terminal domains. Despite confirming an Orai–Orai-dependent specific decline in FRET, a structural basis for this was only inferred in the manuscript by Navarro-Borelly et al. (Navarro-Borelly et al. 2008). A concurrently published paper by Penna et al. (2008) demonstrated that Orai exists as a dimer in the plasma membrane under basal conditions and, when artificially stimulated by the carboxyl terminus of STIM, a four-step photo-bleaching mechanism occurred, suggesting a dimerization of the Orai dimers to form a functional tetrameric channel. Penna and colleagues were able to conclude that the assembly and activation of the functional CRAC are initiated by the C-terminus of the STIM protein (Penna et al. 2008). Earlier studies, using Orai-1 tandem multimers, also demonstrated that functional CRAC are formed by tetrameric assembly of Orai-1 subunits (Ji et al. 2008; Mignen et al. 2008). Furthermore, Muik et al. recently established the importance of a C-terminal coiled-coil motif in Orai-1 as a vital domain for its dynamic coupling to STIM-1 (Muik et al. 2008). A missense mutation in Orai-1 (R91W), a common genetic defect in humans with severe combined immunodeficiency disease (SCID), is known to abolish ICRAC. Navarro-Borelly and co-workers confirmed recent work by other groups (Muik et al. 2008) showing that this mutation had no effect on the interaction and assembly of, or subsequent conformational changes in, STIM-1–Orai-1 and Orai-1–Orai-1 subunits. Similar observations were made by mutations of the selectivity filter in the Orai-1 channel pore region. Confirmation that the observed interactions, redistribution and conformational changes in STIM–Orai and Orai–Orai correlated with functional changes in ICRAC activity using electrophysiological techniques would have further enhanced the significance of the current manuscript by Navarro-Borelly et al. In HEK cells they only demonstrated a functional interaction between over-expressed STIM-1 and Orai-1, which recapitulated the electrophysiological properties of ICRAC (Navarro-Borelly et al. 2008). Taking into consideration the widely debated involvement of TRP channel subunits as SOCs, it remains plausible that Orai could in fact form a heteromeric channel with Orai. Although Navarro-Borelly's investigation did not address this, the exact question was recently addressed by Liao and colleagues (Liao et al. 2008). Using electrophysiological techniques and intracellular Ca2+ imaging they demonstrated a functional interaction between Orai-1 and TRPC subunits under the influence of STIM-1. This potentially adds another level of complexity to this signalling pathway which will need to be fully resolved to understand completely store-depletion-mediated Ca2+ entry into cells. Even though an impressive series of recent publications has strived to identify the signalling mechanisms leading to store-depletion-mediated Ca2+ entry, the precise nature of ICRAC remains elusive. Over-expression of the proposed Ca2+ sensing and pore-forming molecular components, STIM-1 and Orai-1, in the study by Navarro-Borelly et al. has provided crucial information into their interactions in response to store depletion. Future studies are still necessary to identify how the conformational changes lead to gating of CRAC, how the overexpression studies can be translated into a physiological and pathophysiological regulation of CRAC in native cells and it also remains to be determined how TRP channels are involved in SOC. Furthermore, does a mysterious diffusible factor still exist in addition to the STIM–Orai defined pathway of SOC? It is important to determine the interactions of STIM and Orai to understand their contribution to ICRAC; however, other theories remain to be fully substantiated and the physiological and pathophysiological functions to be determined. Does the presence of a diffusible or Ca2+ influx factor still exist in addition to the STIM–Orai defined pathway of SOC (Smani et al. 2004)? Does heterogeneity exist in different cell types in the molecular components or combinations of TRP, Orai and STIM, amongst others, to form functional SOC/CRAC? Does the developmental stage of cells (e.g. stem cells and progenitor cells) and/or cell cycle phase correlate with changes in SOC (Kapur et al. 2007)? The molecular and/or functional composition of SOC may be dynamically and diversely regulated in different cells or in the same cells undergoing physiological and pathophysiological phenotypical changes; it will be important for future studies to consider such questions.