Activation Of Store-operated Calcium Entry Research Articles

Effects of rapamycin-induced oligomerization of parvalbumin, Stim1 and Orai1 in puncta formation

Elevations of cytosolic Ca2+ from the endoplasmic reticulum (ER) regulate a diverse range of cellular processes. When these luminal stores become depleted, the transmembrane ER protein Stim1 oligomerizes and translocates within the ER membrane to puncta junctions to couple with Orai1 channels, activating store-operated calcium entry (SOCE). Stim1 oligomerization and puncta formation have generally been associated with its luminal domains, however, studies have implicated that the cytoplasmic domains may contribute to this oligomerization. Studies have also suggested that intermediate or regulating elements may be required to fine-tune puncta formation and activation of SOCE. Here we made fusion proteins of Stim1 and Orai1 with FRB and FKBP12 domains that associate in the presence of rapamycin. Rapamycin-induced coupling of Stim1 to Stim1, Orai1 to Orai1 and Stim1 to Orai1 was found to be insufficient for puncta formation. Rapamycin was then used to recruit the cytosolic Ca2+ buffer protein parvalbumin (Pav) to Stim1 in order to buffer the local cytosolic Ca2+ near the ER membrane. Interestingly, Pav buffering near the ER caused puncta formation that was indistinguishable from those caused by thapsigargin. Our results suggest that Stim1 oligomerization and puncta formation may be additionally regulated either by local Ca2+ levels near the ER membrane or by as yet unidentified Ca2+-dependent proteins interacting with the cytoplasmic domains of Stim1.

Cytosolic Calcium Dependency of STIM1 De-Oligomerization

Store operated calcium entry (SOCE) is a universal mechanism that cells use to replenish their intracellular calcium stores. SOCE activation is regulated by STIM1, an endoplasmic reticulum (ER) transmembrane protein with calcium binding EF-hand domains protruding into the ER lumen. Upon depletion of ER calcium stores, the dissociation of calcium ions from STIM1 EF-hands triggers a conformational change that leads to the formation of STIM1 oligomers, which translocate to the plasma membrane to bind and activate calcium channels of the Orai family.

SERAF, a Novel Regulator of Store Operated Calcium Entry

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular calcium stores and execute a wide range of specialized activities. In recent years tremendous progress has been made in understanding the nature of the molecular components and events that govern SOCE activation. STIM and Orai were identified as the essential components able to reconstitute Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SERAF, a novel regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis, combined with electrophysiological, biochemical and imaging techniques; we show that SERAF is an endoplasmic reticulum membrane protein that associates with STIM to facilitate SOCE inactivation following calcium refilling. SERAF, therefore, plays a key role in shaping cytosolic Ca2+ signals and determining the content of the major intracellular calcium stores, role that is likely to be important for protecting cells from calcium overfilling.

Biophysical and Pharmacological Characterisation of Calcium Channels in Human Mast Cells

The best characterised calcium channel in immune cells is the store-operated calcium release activated calcium channel (CRAC); studies in heterologous expression systems and knockout mice have identified Orai and its regulator STIM1, an ER-resident calcium sensor, as the molecular constituents of CRAC. Members of the TRPC family are also likely to contribute to store-operated calcium entry (SOCE) in immune cells. The expression of Orai and TRPC channels in primary human mast cells has not yet been investigated, but is of key importance given the critical role these cells play in the pathophysiology of type-1 hypersensitivity reactions. Allergic activation occurs following cross-linking of the FcεRI receptor by antigen-IgE complexes that, via PLC-γ signalling, leads to robust activation of SOCE. The resulting calcium signal stimulates secretion and production of pro-inflammatory mediators. The aim of this study was to characterize the biophysical and pharmacological properties of CRAC channels in human lung mast cells (HLMC) following SOCE. RT-PCR revealed mRNA expression for Orai1, Orai2, STIM1 and STIM2 but not Orai3 in HLMCs. Whole-cell patch clamp recordings of isolated, cultured HLMCs were carried out using a Cs-based internal solution, with added IP3 and BAPTA to activate SOCE. These conditions activated an inwardly rectifying current with Erev= 18±4mV (n=6), a value less positive than expected for a pure Orai/CRAC current. However, the current was potentiated in divalent-free solution 563.7±6% (n=6), as expected for Orai/CRAC currents, and was significantly attenuated 51.4±8% (n=6), by the Orai-specific inhibitor Synta66 (10μM) (Ng et al. 2008., Di Sabatino et al. 2009). Calcium imaging in HLMCs loaded with fura-2 AM showed that Synta66 also attenuated anti-IgE induced calcium signaling by 62.2%(n=14). Taken together the data suggest that both Orai and TRPC channels contribute to SOCE in HLMCs.

Remodelling of the endoplasmic reticulum during store‐operated calcium entry

SOCE (store-operated calcium entry) is a ubiquitous cellular mechanism linking the calcium depletion of the ER (endoplasmic reticulum) to the activation of PM (plasma membrane) Ca2+-permeable channels. The activation of SOCE channels favours the entry of extracellular Ca2+ into the cytosol, thereby promoting the refilling of the depleted ER Ca2+ stores as well as the generation of long-lasting calcium signals. The molecules that govern SOCE activation comprise ER Ca2+ sensors [STIM1 (stromal interaction molecule 1) and STIM2], PM Ca2+-permeable channels {Orai and TRPC [TRP (transient receptor potential) canonical]} and regulatory Ca2+-sensitive cytosolic proteins {CRACR2 [CRAC (Ca2+ release-activated Ca2+ current) regulator 2]}. Upon Ca2+ depletion of the ER, STIM molecules move towards the PM to bind and activate Orai or TRPC channels, initiating calcium entry and store refilling. This molecular rearrangement is accompanied by the formation of specialized compartments derived from the ER, the pre-cER (cortical ER) and cER. The pre-cER appears on the electron microscope as thin ER tubules enriched in STIM1 that extend along microtubules and that are devoid of contacts with the PM. The cER is located in immediate proximity to the PM and comprises thinner sections enriched in STIM1 and devoid of chaperones that might be dedicated to calcium signalling. Here, we review the molecular interactions and the morphological changes in ER structure that occur during the SOCE process.

Possible Role of BDNF-Induced Microglial Intracellular Ca<sup>2+</sup> Elevation in the Pathophysiology of Neuropsychiatric Disorders

Microglia are intrinsic immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO) and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions, such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. We have recently reported that BDNF induces a sustained increase in [Ca(2+)]i through binding with the truncated TrkB receptor, resulting in activation of the PLC pathway and store-operated calcium entry (SOCE) in rodent microglial cells. Sustained activation of SOCE, possibly mediated by TRP channels, occurred after brief BDNF application and contributed to the maintenance of sustained [Ca(2+)]i elevation. Pretreatment with BDNF significantly suppressed the release of NO from activated microglia. Additionally, selective serotonin reuptake inhibitors (SSRIs), including paroxetine or sertraline, potentiated the BDNF-induced increase in [Ca(2+)]i in rodent microglial cells This article provides a review of recent findings on the role of BDNF in the pathophysiology of neuropsychiatric disorders, especially by focusing on its effect on intracellular Ca(2+) signaling in microglial cells.

STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

Effect of cell swelling on ER/PM junctional interactions and channel assembly involved in SOCE

Store-operated calcium entry (SOCE) regulates critical cellular functions and is determined by precise ER/plasma membrane (PM) junctional interactions. Here we have assessed the effect of hypotonic cell volume increase on SOCE in a salivary gland epithelial cell line (HSG). Thapsigargin (Tg) activated a 2APB- and 1μM Gd3+-sensitive, inwardly rectifying, cation current, ISOC, while hypotonic solution (150mOsm) induced cell swelling and activated an outwardly rectifying cation current that was blocked by 100μM Gd3+ but not by 2APB. HTS addition before or after Tg attenuated the sensitivity of Ca2+ influx to 2APB and 1μM Gd3+. After HTS-induced volume increase, while stimulation of cells with Tg resulted in intracellular Ca2+ release without Ca2+ influx, stimulation with CCh caused neither internal Ca2+ release nor Ca2+ influx. Importantly, HTS caused the ER to recede from the plasma membrane which prevented Tg-stimulated clustering of STIM1 in the ER/PM region and association of STIM1 with TRPC1 and Orai1. Disruption of SOCE was dependent on the level of hypotonic stress as 225mOsm HTS induced relatively less cell swelling or disruption of SOCE. These results demonstrate that epithelial cells can tolerate small increases (up to 5%) in cell volume while larger increases lead to disruption of ER–PM interactions that are critical for activation of SOCE. We suggest that loss of SOCE could impact cell function and contribute to the deleterious effects of severe hypotonic stress.

Passive Activation of Store-Operated Ca2+ Entry in Myotubes Depends on the Rate of RyR1-Dependent Ca2+ Leak

In spite of extensive studies of store operated calcium entry (SOCE), the detailed mechanism of SOCE activation in skeletal muscle remains largely unknown. We recently reported that STIM1-Orai1 coupling is required for SOCE activation in myotubes. However, other proteins that control sarcoplasmic reticulum (SR) Ca2+ content may also contribute to SOCE activation. We hypothesized that passive SOCE activation in skeletal muscle depends on the rate of SR Ca2+ leak through the type 1 ryanodine receptor (RyR1). To test this hypothesis, we conducted a series of whole-cell patch-clamp measurements of SOCE current (ISOC) in myotubes obtained from normal and RyR1-null (dyspedic) mice. Myotubes were bathed in external solution containing (in mM): 138 TEA-methanesulfonate, 10 CaCl2, 10 HEPES, 1 MgCl2, 0.1 nifedipine, pH 7.4. The internal patch pipette solution contained (in mM): 140 mM Cs-methanesulfonate, 10 HEPES, 20 Na-EGTA, 4 MgCl2, pH7.4. SOCE was activated by passive SR Ca2+ depletion following intracellular dialysis with 20 mM EGTA. ISOC in myotubes exhibited many hallmarks of SOCE including strong inward rectification and inhibition by La3+, Gd3+, BTP-2, and 2-APB. ISOC current density at −80 mV was significantly (p<0.01) larger in normal myotubes (1.05 ± 0.09 pA/pF, n = 33) compared to that from dyspedic myotubes (0.74 ± 0.07 pA/pF, n = 18). Moreover, the speed of ISOC activation was slower in dyspedic myotubes. Specifically, the time to 10%, 50%, and 90% maximal activation were 4.95 ± 0.65 s, 15.3 ± 1.4 s, and 53.9 ± 7.5 s (n = 19), respectively, in normal myotubes and 67.5 ± 11.1 s, 114 ± 13 s, and 159 ± 17 s (n = 18), respectively, in dyspedic myotubes. These results indicate that RyR1 Ca2+ leak promotes passive SOCE activation.

Ryanodine Receptors Control Cytosolic Calcium Elevation Following Activation of Store-Operated Calcium Entry in Activated but not Resting Human T Lymphocytes

Previously we have shown that in Jurkat T lymphocytes, the ryanodine (RyR) receptors are activated by store-operated Ca2+ entry (SOCE) and that inhibition of RyR significantly reduced elevation in intracellular Ca2+ concentration ([Ca2+]i) following SOCE. Because Jurkat T cells differ from normal human T cells, we explored contribution of RyR into Ca2+ signaling in two functional human T lymphocyte subsets: resting and activated. Resting T cells were isolated from the peripheral blood of healthy humans and activated in vitro using anti-CD3 and anti-CD28 antibodies. Assessing the [Ca2+]i dynamics in activated T cells using fura-2, a Ca2+ indicator, revealed that RyR blockers ryanodine (Ry) and dantrolene (Da) significantly reduced Ca2+ elevation upon SOCE activation, while increasing Ca2+ content within the store, which is consistent with our previous findings in Jurkat T cells. In contrast, in resting T cells neither Ry nor Da affected [Ca2+]i elevation upon SOCE activation at physiological concentration (2 mM) of extracellular Ca2+. However, the inhibitory effects of RyR blockers were observed in resting T cells in the presence of the elevated extracellular Ca2+concentration (10 mM). Using Mn2+ quench of fura 2 fluorescence approach we further explored whether inhibition of [Ca2+]i elevation in the presence of RyR blockers could be attributed to termination of SOCE due to the Ca2+ accumulation within the store. We found that rates of Mn2+ quench were identical in the presence and absence of RyR blockers, indicating that within a given timeframe enhanced Ca2+ accumulation within the store did not affect SOCE. We conclude that in activated human T cells Ry-sensitive store serves as an intermediate compartment for SOCE and that RyR controls [Ca2+]i dynamics by regulating [Ca2+]i release from the store.

Brain-Derived Neurotrophic Factor Induces Sustained Elevation of Intracellular Ca2+ in Rodent Microglia

Microglia are intrinsic immune cells that release factors, including proinflammatory cytokines, NO, and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions, such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we observed that BDNF induced a sustained increase in [Ca(2+)]i through binding with the truncated tropomyosin-related kinase B receptor, resulting in activation of the PLC pathway and store-operated calcium entry in rodent microglial cells. RT-PCR and immunocytochemical techniques revealed that truncated tropomyosin-related kinase B-T1 receptors were highly expressed in rodent microglial cells. Sustained activation of store-operated calcium entry occurred after brief BDNF application and contributed to the maintenance of sustained [Ca(2+)]i elevation. Pretreatment with BDNF significantly suppressed the release of NO from activated microglia. Additionally, pretreatment of BDNF suppressed the IFN-gamma-induced increase in [Ca(2+)]i, along with a rise in basal levels of [Ca(2+)]i in rodent microglial cells. We show direct evidence that rodent microglial cells are able to respond to BDNF, which may be important for the regulation of inflammatory responses, and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

IP 3 receptor subtype-dependent activation of store-operated calcium entry through I CRAC

The store-operated, calcium release-activated calcium current I CRAC is activated by the depletion of inositol 1,4,5-trisphosphate (IP 3)-sensitive stores. The significantly different dose–response relationships of IP 3-mediated Ca 2+ release and CRAC channel activation indicate that I CRAC is activated by a functionally, and possibly physically, distinct sub-compartment of the endoplasmic reticulum (ER), the so-called CRAC store. Vertebrate genomes contain three IP 3 receptor (IP 3R) genes and most cells express at least two subtypes, but the functional relevance of various IP 3R subtypes with respect to store-operated Ca 2+ entry is completely unknown. We here demonstrate in avian B cells (chicken DT40) that IP 3R type II and type III participate in IP 3-induced activation of I CRAC, but IP 3R type I does not. This suggests that the expression pattern of IP 3R contributes to the formation of specialized CRAC stores in B cells.

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca(2+)](i) to a maximum of 27.09 +/- 4.35 nM from the resting value. The activation time to reach 10-90% of the maximum amplitude was 112 +/- 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 +/- 23 s and raised the [Ca(2+)](i )to 129.50 +/- 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca(2+)](i) to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca(2+)](i) but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.

A key role for reverse Na +/Ca 2+ exchange influenced by the actin cytoskeleton in store-operated Ca 2+ entry in human platelets: Evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP 3 receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca 2+ but not Mn 2+ or Na + entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca 2+ but not Mn 2+ or Na + entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na +/Ca 2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca 2+ but not Mn 2+ or Na + entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca 2+ permeable pathways activated following Ca 2+ store depletion in human platelets: A Ca 2+-permeable, hTRPC1-containing SOC and reverse Na +/Ca 2+ exchange, which is activated following Na + entry through the SOC and requires a functional actin cytoskeleton.

TRPC1: The link between functionally distinct store-operated calcium channels

Although store-operated calcium entry (SOCE) was identified more that two decades ago, understanding the molecular mechanisms that regulate and mediate this process continue to pose a major challenge to investigators in this field. Thus, there has been major focus on determining which of the models proposed for this mechanism is valid and conclusively establishing the components of the store-operated calcium (SOC) channel(s). The transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the elusive store-operated Ca 2+ entry channel. While all TRPCs are activated in response to agonist-stimulated phosphatidylinositol 4,5, bisphosphate (PIP 2) hydrolysis, only some display store-dependent regulation. TRPC1 is currently the strongest candidate component of SOC and is shown to contribute to SOCE in many cell types. Heteromeric interactions of TRPC1 with other TRPCs generate diverse SOC channels. Recent studies have revealed novel components of SOCE, namely the stromal interacting molecule (STIM) and Orai proteins. While STIM1 has been suggested to be the ER-Ca 2+ sensor protein relaying the signal to the plasma membrane for activation of SOCE, Orai1 is reported to be the pore-forming component of CRAC channel that mediates SOCE in T-lymphocytes and other hematopoetic cells. Several studies now demonstrate that TRPC1 also associates with STIM1 suggesting that SOC and CRAC channels are regulated by similar molecular components. Interestingly, TRPC1 is also associated with Orai1 and a TRPC1-Orai1-STIM1 ternary complex contributes to SOC channel function. This review will focus on the diverse SOC channels formed by TRPC1 and the suggestion that TRPC1 might serve as a molecular link that determines their regulation by store-depletion.

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca 2+ entry activated in human platelets by thapsigargin-evoked Ca 2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC- l-leucyl- l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca 2+ store depletion even in the absence of a rise in cytosolic [Ca 2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca 2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca 2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca 2+ entry pathways.

A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets

AbstractStore-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in platelets and other cells. De novo conformational coupling between elements in the plasma membrane and Ca2+ stores, where the actin cytoskeleton plays an important regulatory role, has been proposed as the most likely mechanism to activate SOCE in platelets. Here we have examined for the first time changes in platelet F-actin levels on a subsecond time scale. Using stopped-flow fluorimetry and a quenched-flow approach, we provide evidence for the involvement of cofilin in actin filament reorganization and SOCE in platelets. Thrombin (0.1 U/mL) evoked an initial decrease in F-actin that commenced within 0.1 second and reached a minimum 0.9 second after stimulation, prior to the activation of SOCE. F-actin then increased, exceeding basal levels approximately 2.5 seconds after stimulation. Thrombin also induced cofilin dephosphorylation and activation, which paralleled the changes observed in F-actin, and rapid Btk activation. Inhibition of cofilin dephosphorylation by LFM-A13 resulted in the loss of net actin depolymerization and an increased delay in SOCE initiation. These results suggest that cofilin is important for the rapid actin remodeling necessary for the activation of SOCE in platelets through de novo conformational coupling.

The Foot Structure from the Type 1 Ryanodine Receptor Is Required for Functional Coupling to Store-operated Channels

In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

Calcium Channel Mechanisms in Lacrimal Acinar Cells and other Non-Excitable Cells

Although store-operated calcium entry (SOCE) was identified more that two decades ago, understanding the molecular mechanisms that regulate and mediate this process continue to pose a major challenge to investigators in this field. Thus, there has been major focus on determining which of the models proposed for this mechanism is valid and conclusively establishing the components of the store-operated calcium (SOC) channel(s). The transient receptor potential canonical (TRPC) proteins have been suggested as candidate components of the elusive store-operated Ca2+ entry channel. While all TRPCs are activated in response to agonist-stimulated phosphatidylinositol 4,5, bisphosphate (PIP2) hydrolysis, only some display store-dependent regulation. TRPC1 is currently the strongest candidate component of SOC and is shown to contribute to SOCE in many cell types. Heteromeric interactions of TRPC1 with other TRPCs generate diverse SOC channels. Recent studies have revealed novel components of SOCE, namely the stromal interacting molecule (STIM) and Orai proteins. While STIM1 has been suggested to be the ER-Ca2+ sensor protein relaying the signal to the plasma membrane for activation of SOCE, Orai1 is reported to be the pore-forming component of CRAC channel that mediates SOCE in T-lymphocytes and other hematopoetic cells. Several studies now demonstrate that TRPC1 also associates with STIM1 suggesting that SOC and CRAC channels are regulated by similar molecular components. Interestingly, TRPC1 is also associated with Orai1 and a TRPC1-Orai1-STIM1 ternary complex contributes to SOC channel function. This review will focus on the diverse SOC channels formed by TRPC1 and the suggestion that TRPC1 might serve as a molecular link that determines their regulation by store-depletion.

Store-operated calcium entry and increased endothelial cell permeability.

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.