ER-PM Junctions Research Articles

Trafficking and Regulation of Orai1 in Mammalian Cells

Store-operated Ca2+ entry (SOCE) is activated by endoplasmic reticulum Ca2+ store depletion and is a major Ca2+ influx pathway in non-excitable cells. It is widely accepted that Orai1 protein in the plasma membrane (PM) is the channel that mediates SOCE in many cell types. Orai1 is activated when STIM1, an ER transmembrane protein, senses a decrease in the ER Ca2+ concentration and redistributes to ER-PM junctions, where it recruits Orai1 in coincident puncta. We have recently shown that at rest Orai1 actively recycles between an endosomal compartment and the cell membrane in Xenopus laevis oocytes and that ER Ca2+ store depletion translocates most of the Orai1 to the PM. We have also shown that during oocyte maturation Orai1 is internalized resulting in the absence of SOCE in the Xenopus egg. In mammalian cells, mitosis is the only know physiological situation where SOCE is inhibited but the underlying mechanism is not fully understood. In this study we investigated the trafficking of Orai1 in CHO cells that express a tagged Orai1. We also looked at the distribution of Orai1 during mitosis. We show that at steady state about 40% of the total Orai1 pool is at the PM whilst the remaining 60% localizes intracellulary, suggesting that Orai1 constitutively recycles between the two compartments. After the depletion of the ER Ca2+ stores the distribution of Orai1 shifts drastically with now most of the protein localized at the PM. Moreover, we show that the rate of Orai1 exocytosis is 3 fold faster after the depletion of the ER Ca2+ stores. During mitosis the total expression of Orai1 is reduced and the fraction of plasma membrane Orai1 tend to be less than in interphase cells. A fraction of the mitotic cells show no Orai1 expression at all. Interestingly, the reduction of Orai1 expression is reversed by inhibiting the proteosome complex, suggesting that Orai1 is degraded during mitosis.

Stoichiometric requirements for trapping and gating of Ca 2+ release-activated Ca 2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

Mitofusin 2 Regulates STIM1 Migration from the Ca2+ Store to the Plasma Membrane in Cells with Depolarized Mitochondria

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.

The Facultative Role of Lipid Rafts and Membrane Microdomains in Controlling the Assembly of the Store-Operated Channel Complex

Calcium influx is an important factor in a wide variety of cellular functions. The signal initiating calcium influx may originate from the activation of G-protein coupled receptors, or arise from inside the cell, via maneuvers that can lead to depletion of intracellular calcium storage compartments. While the role of members from the Transient Receptor Potential Cation (TRPC) family of channels as receptor-operated channels (ROC) is well established and supported by abundant studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We have found that the association of STIM1 to TRPC1 favors the insertion of this channel into lipid rafts, where TRPC1 functions as a SOC in association to Orai channels forming a complex we have named SOCIC (store-operated calcium influx complex). In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel imaging method implemented for the present study based on multicolor LightGuide Total Internal Reflection Fluorescence Microscopy (LG-TIRFM), illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1-Orai complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains (lipid rafts) and ER-PM junctions in modulating TRPC1 channel function. Using LG-TIRFM, we identified new partners of the SOCIC, and studied the dynamics of SOCIC assembly and disassembly in real time.

STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1

Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

Essential Roles for Coiled-coil Domains in STIM1 Oligomerization and CRAC Channel Activation

The calcium release-activated calcium (CRAC) channel is activated by depletion of Ca2+ from the ER. Store depletion causes the ER Ca2+ sensor, STIM1, to translocate to sites of close ER-plasma membrane apposition, where it interacts with Orai1, the pore-forming subunit of the CRAC channel and activates Ca2+ entry. STIM1 self-associates in resting cells (Baba et al., PNAS 103:16704, 2006) and further oligomerizes after store depletion (Liou et al., PNAS 104:9301, 2007), an event that triggers the self-assembly and activation of STIM1-Orai1 clusters at ER-PM junctions (Luik et al., Nature 454:538, 2008). STIM1 has several protein interaction domains, including a lumenal sterile alpha motif (SAM) and two putative cytosolic coiled-coil regions. The isolated lumenal EF hand - SAM region is known to oligomerize upon Ca2+ removal in vitro (Stathopulos et al., JBC 281:35855, 2006), but the roles of the coiled-coil domains in the functions of STIM1 in situ are not as well understood. Using fluorescence recovery after photobleaching, co-immunoprecipitation, and blue native PAGE analysis on truncated mutants of STIM1 we show that the two coiled-coil domains of STIM1 affect STIM1 oligomerization in different ways. The ER-proximal coiled-coil is sufficient for the self-association of STIM1 in resting cells but does not by itself support oligomerization in response to store depletion. The distal coiled-coil is required for depletion-induced oligomerization. Mutation of specific residues within the predicted hydrophobic interface of the distal coiled-coil prevents the formation of STIM1 puncta and the activation of CRAC channels. These results reveal an essential role for the distal coiled-coil of STIM1 in the oligomerization step that controls store-operated Ca2+ entry.

Store‐operated channels: mechanisms and function

For many years, how Ca2+ entered non-excitable cells remained enigmatic. There was a suspicion that Ca2+ channels were involved, but what these were and how they were gated was unclear. By contrast, not only were the Ca2+ channels expressed in excitable cells well defined electrophysiologically but their subunit composition and even structure–function profile had been teased apart. This imbalance began to change in 1986, when a landmark paper by James Putney let to a paradigmatic shift in our understanding of Ca2+ entry in non-excitable cells (Putney, 1986). In that classic review, Putney proposed the concept of capacitative (now called store-operated) Ca2+ influx. In its most fundamental form, emptying of agonist-sensitive intracellular Ca2+ stores led to the opening of plasma membrane Ca2+ channels. Although this model was largely overlooked in its early days, two subsequent discoveries established its importance in cell physiology. First was the finding that thapsigargin, a sequiterpene lactone occurring naturally in plants, blocked Ca2+ pumps on the stores and therefore depleted them of Ca2+. Crucially, after store depletion to thapsigargin, Ca2+ influx occurred (Takemura et al. 1989). This supported the key tenet of the store-operated model. Second and more crucially perhaps, an elegant series of patch clamp experiments by Hoth & Penner (1992) revealed the presence of a Ca2+-selective current that was activated on emptying intracellular stores. The underlying channels, which they called Ca2+ release-activated Ca2+ (CRAC) channels, could be activated regardless of how stores were emptied and provided a key electrophysiological signature for store-operated Ca2+ entry. CRAC channels have several intriguing features for ion channels including a tiny single channel conductance and exquisite selectivity for Ca2+ (reviewed in Parekh & Putney, 2005), but for many years the vexing question that engaged most researchers was the molecular basis of store-operated entry. How is Ca2+ sensed within the endoplasmic reticular Ca2+ store? How is information sent from the store to the plasma membrane? What is the molecular identity of the CRAC channel? Our understanding of store-operated Ca2+ has increased over the past three years with the identification, using primarily siRNA technology, of two key components: STIM1 and Orai1–3 (reviewed in Lewis, 2007). The pace has been breathtaking, and a picture has emerged based on solid experimental evidence from several laboratories. STIM1 is the Ca2+ sensor and Orai1 is all or part of the CRAC channel pore. Upon store depletion, STIM1 molecules migrate to form discrete puncta just below the plasma membrane where they recruit and activate Orai1. At a recent meeting of the Biophysical Society in Long Beach, California, The Journal of Physiology held a symposium entitled ‘Store-operated channels: mechanism and function’. This provided an opportunity to reflect on the recent rapid progress in the field and identify new avenues for research. Victoria Bolotina (Boston) described her recent work suggesting that a small diffusible messenger called Ca2+ influx factor (CIF) linked STIM1 to Orai1 channel activation and that STIM1 might function as an enzyme that produces CIF (Bolotina, 2008). Richard Lewis (Stanford) described how STIM1 movement to specialized ER–PM junctions occurred and that STIM1 oligomerization was sufficient to activate CRAC channels. Anant Parekh (Oxford) showed data documenting that local Ca2+ rises near CRAC channels could drive the synthesis of intra- and intercellular signalling molecules and reported the underlying signalling cascades (Parekh, 2008). Reinhold Penner (Hawaii) compared the properties of Orai1, 2 and 3 and found striking differences in how the pharmacological tool 2-APB interacted with the channels, revealing differences in channel gating (Peinelt et al. 2008). Finally, James Putney (NIEHS, Research Triangle Park) provided convincing evidence that Ca2+ influx through CRAC channels supported intracellular Ca2+ oscillations in response to low concentrations of agonist and presented data showing oscillatory movement of STIM1 up to the plasma membrane in parallel with the Ca2+ spikes (Putney & Bird, 2008). The talks presented at the symposium have been included as a series of Symposium Reviews and related research papers in this issue. In short, the symposium showed how far the store-operated channel field has advanced in a relatively short time. No doubt, with the approaches and techniques now available, new findings and unexpected twists will be revealed in the future.

STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: Moving TRPC1 in and out of lipid rafts

While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

STIM1 Knockdown Reveals That Store-operated Ca2+ Channels Located Close to Sarco/Endoplasmic Ca2+ ATPases (SERCA) Pumps Silently Refill the Endoplasmic Reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.