Edited by: Francesco Moccia, University of Pavia, Italy
Reviewed by: Menahem Segal, Weizmann Institute of Science, Israel; Gaiti Hasan, National Centre for Biological Sciences, India
*Correspondence: Joanna Gruszczynska-Biegala
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The process of store-operated calcium entry (SOCE) leads to refilling the endoplasmic reticulum (ER) with calcium ions (Ca2+) after their release into the cytoplasm. Interactions between (ER)-located Ca2+ sensors (stromal interaction molecule 1 [STIM1] and STIM2) and plasma membrane-located Ca2+ channel-forming protein (Orai1) underlie SOCE and are well described in non-excitable cells. In neurons, however, SOCE appears to be more complex because of the importance of Ca2+ influx via voltage-gated or ionotropic receptor-operated Ca2+ channels. We found that the SOCE inhibitors ML-9 and SKF96365 reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i amplitude by 80% and 53%, respectively. To assess the possible involvement of AMPA receptors (AMPARs) in SOCE, we used their specific inhibitors. As estimated by Fura-2 acetoxymethyl (AM) single-cell Ca2+ measurements in the presence of CNQX or NBQX, thapsigargin (TG)-induced Ca2+ influx decreased 2.2 or 3.7 times, respectively. These results suggest that under experimental conditions of SOCE when Ca2+ stores are depleted, Ca2+ can enter neurons also through AMPARs. Using specific antibodies against STIM proteins or GluA1/GluA2 AMPAR subunits, co-immunoprecipitation assays indicated that when Ca2+ levels are low in the neuronal ER, a physical association occurs between endogenous STIM proteins and endogenous AMPAR receptors. Altogether, our data suggest that STIM proteins in neurons can control AMPA-induced Ca2+ entry as a part of the mechanism of SOCE.
Neuronal Ca2+ homeostasis is precisely regulated by complex mechanisms (Berridge,
Recent studies found that STIM1, in addition to Orai activation, inhibits Ca2+ entry that is mediated by T- and L-type channels in excitable cells (Park et al.,
AMPARs are tetramers that are composed of homologous glutamate GluA1-GluA4 subunits in various combinations (Bigge,
In the present study, we analyzed the possible link between Ca2+ entry via AMPARs and SOCE in neurons. We found that SOCE was blocked by AMPAR antagonists and that SOCE inhibition decreased [Ca2+]i rise that was induced by AMPAR agonist. These results, together with co-immunoprecipitation assays that indicated interactions between endogenous STIMs and AMPAR subunits, indicate the possible role of STIMs in AMPA-induced [Ca2+]i changes. These observations elucidate the role of STIM proteins in the influx of Ca2+ via channels other than Orai and TRPs.
Cortical neuronal cultures were prepared from embryonic day 19 (E19) Wistar rat brains. Pregnant female Wistar rats were provided by the Animal House of the Mossakowski Medical Research Centre, Polish Academy of Sciences (Warsaw, Poland). Animal care was in accordance with the European Communities Council Directive (86/609/EEC). The experimental procedures were approved by the Local Commission for the Ethics of Animal Experimentation no. 1 in Warsaw. Brains were removed from rat embryos and collected in cold Hanks solution supplemented with 15 mM HEPES buffer and penicillin/streptomycin. The cortices were isolated, rinsed three times in cold Hanks solution, and treated with trypsin for 35 min. The tissue was then rinsed in warm Hanks solution, washed three times, and dissociated by pipetting. For Ca2+ measurements, primary cortical neurons were plated at a density of 7 × 104 cells/well on eight-well PDL-laminin-precoated chamber slides (BioCoat). For the co-immunoprecipitation assays and Western blot, neurons were seeded on poly-D-lysine-precoated BioCoat plastic Petri dishes (BD Biosciences Discovery Labware) at a density of 7 × 106 cells/plate. Neurons were grown in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine (Sigma), 12.5 μM glutamate (Sigma), and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco). Cultures were maintained at 37°C in a humidified 5% CO2/95% air atmosphere. Every 3–4 days, half of the conditioned medium was removed and replaced by fresh growth medium. The experiments were performed on 15-day-old cultures (co-immunoprecipitation assay, Western blot) or 16- to 17-day-old cultures (Ca2+ measurements).
To make the glial cultures, a cortical cell suspension was plated on poly-D-lysine-precoated BioCoat plastic Petri dishes (BD Biosciences Discovery Labware) and grown in Dulbecco’s Modified Eagle Medium (DMEM) that contained 10% fetal bovine serum and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco). The cells were grown to confluence (after ~1 week) and were replated to avoid non-glial contamination. The medium was replaced once every 3–4 days. For Western blot, the cells were cultured for 15 days prior to the experiments.
HeLa cells were grown in DMEM that contained 10% fetal bovine serum and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco) at 37°C in a 5% CO2 atmosphere.
Single-cell Ca2+ levels in cortical neurons were recorded using the ratiometric Ca2+ indicator dye Fura-2 acetoxymethyl ester (Fura-2 AM). Cells were grown on eight-well chamber slides and loaded with 2 μM Fura-2 AM for 30 min at 37°C in Hanks Balanced Salt Solution (HBSS) that contained 145 mM NaCl, 5 mM KCl, 0.75 mM Na2HPO4, 10 mM glucose, 10 mM HEPES (pH 7.4) and 1 mM MgCl2 supplemented with 2 mM CaCl2 at 37°C (high Ca2+ medium) and then rinsed and left undisturbed for 30 min at 37°C to allow for de-esterification. Measurements of intracellular Ca2+ levels were performed every 1 s at 37°C using an Olympus Scan∧R&Cell∧R imaging system that consisted of an IX81 microscope (Olympus, Tokyo, Japan), 10× 0.40 NA UPlanS Apo objective (Olympus, Tokyo, Japan), and Hamamatsu EM-CCD C9100-02 camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Changes in intracellular Ca2+ concentration ([Ca2+]i) in individual neuronal cell bodies are expressed as the F340/F380 ratio after subtracting background fluorescence. This ratio represents the emission intensities at 510 nm obtained after excitation at 340 and 380 nm. The low Ca2+ medium (Ca2+-free solution) contained 0.5 mM ethylene glycol tetraacetic acid (EGTA) in the standard buffer. At the end of the experiments, 50 mM KCl in the presence of 2 mM CaCl2 was added to assess which cells were neurons (Figures
For the co-immunoprecipitation of endogenous STIM1, STIM2, GluA1, and GluA2 proteins, 15-day-old primary cortical neurons that were grown on Petri dishes were washed with cold phosphate-buffered saline (PBS), scraped and centrifuged at 1000× g for 5 min at 4°C. The pellet was suspended in lysate buffer, pH 7.5, that contained 50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride supplemented with complete Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche) and lysed with an insulin syringe (18×). After incubation for 2 h on ice and centrifugation at 15,000× g (neuronal lysates) for 20 min at 4°C, cleared lysates were pre-incubated with 30 μl of washed protein A-Sepharose (Roche) for 3 h at 4°C. Sepharose resin and bound components were recovered by centrifugation at 15,000× g for 20 min at 4°C. Precleared lysates were subsequently incubated overnight at 4°C on a rocking platform with 30 μl of A-Sepharose that was pre-incubated earlier for 3 h with 3 μg of antibody (anti-STIM1, BD Transduction Laboratories or ProSci Inc., Poway, CA, USA; anti-STIM2, Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-GluA1, Abcam, UK; anti-GluA2, ProteinTech Group or Alomone Labs). As a negative control when indicated, lysates were omitted or were incubated with anti-Flag (Sigma) or anti-IgG antibody (Sigma). Precipitated samples were then washed three times with repeated centrifugation, eluted in 50 μl of 2× Laemmli Buffer. Protein extracts were resolved by 6% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a Protran nitrocellulose membrane (Whatman), and blocked for 2 h at room temperature in TBST (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) plus 5% dry non-fat milk. Nitrocellulose sheets were then incubated in blocking solution with primary antibodies against STIM1 (1:200, ProteinTech Group), STIM2 (1:100, Alomone Labs), GluA1 (1:400, Merck Millipore) and GluA2 (1:300, ProteinTech Group) at 4°C overnight. The appropriate horseradish peroxidase-conjugated secondary antibody IgG (Sigma) was added at a dilution of 1:10,000 for 1 h. The peroxidase was detected with a Chemiluminescent Substrate Reagent Kit (Novex ECL, Invitrogen).
On Western blot containing glial cells extracts, the optical density of the bands was estimated using a GS-800 Calibrated Densitometer and Quantity One software (Bio-Rad). GAPDH was run to normalize the protein loading.
The statistical analysis was performed using Prism 5.02 software (GraphPad, San Diego, CA, USA). All of the data are expressed as mean ± standard error of the mean (SEM), and differences were considered significant at
The SOCE inhibitor ML9 was used to determine whether AMPA-induced [Ca2+]i responses are affected. Rat cortical neurons were loaded with the Fura-2 AM Ca2+ indicator in 2 mM CaCl2-containing medium, and the Ca2+ signal was recorded. The cells were then treated with 2 mM Ca2+ medium supplemented with 100 μM AMPA in the absence or presence of 100 μM ML-9. After the addition of AMPA, an increase in [Ca2+]i changes was observed in both neuronal cultures (Figure
We next sought to determine whether AMPA-induced elevation in [Ca2+]i in cortical neurons involves other receptors or channels. Neither the VGCC blocker nimodipine (NM; Figures
Since the SOCE inhibitors blocked AMPA-induced [Ca2+]i rise, we next investigated whether AMPAR activity participates in SOCE. We used the potent AMPAR antagonists CNQX and NBQX. Ca2+ recordings were performed in rat cortical neurons. Cells were loaded with the Fura-2 Ca2+ indicator in 2 mM CaCl2-containing medium and then incubated with 0.5 mM EGTA medium to initiate the measurements. The neuronal cultures were then treated with thapsigargin (TG) and either NBQX or CNQX. After 5 min, a medium with 2 mM CaCl2 was added to induce SOCE in the presence of 30 μM NBQX or 30 μM CNQX. To distinguish neurons from other cells that were present in the culture, KCl was added at the end of the experiments, and the Ca2+ signal was recorded (data not shown). Only cells that responded with fast and transient increases in Ca2+ were considered neurons (Figure
In the above experiments, cells in culture that did not respond to KCl were considered glial cells. The data that were collected for these cells were also analyzed (Figure
We next investigated whether this difference can be explained by the expression of different AMPAR subunits in neurons and glia. To examine the protein expression of the AMPAR GluA1 and GluA2 subunits, Western blot was performed using lysates from cultures of neuronal and glial cells that were grown separately. Both GluA1 and GluA2 were detected in neurons. In glial cells, they markedly decreased or were barely detectable (Figure
To exclude the possibility that NBQX inhibits SOCE by interacting with the STIM/Orai complex, we tested its effect in HeLa cells, which lack AMPARs. The treatment of HeLa cells with 30 μM NBQX did not affect the average peak of TG-induced Ca2+ release (Figure
The data above suggest a relationship between AMPARs and SOCE. To confirm the observed participation of AMPARs in [Ca2+]i elevation under SOCE conditions, we evaluated whether endogenous STIM proteins interact with endogenous AMPAR subunits. We performed co-immunoprecipitation experiments using extracts of cultures of rat cortical neurons that were treated with TG to induce SOCE. Specific STIM1, STIM2, GluA1, and GluA2 antibodies were used, and the immunoprecipitates were analyzed by Western blot. Anti-Flag antibodies and IgG were used to identify possible nonspecific interactions. When the neuronal lysates were immunoprecipitated with the anti-STIM1 antibody, the bands of GluA1 (Figure
In reverse experiments, in which neuronal lysates were immunoprecipitated with anti-GluA1 antibody (Figures
The major Ca2+ influx pathway in neurons is through VGCCs and ionotropic glutamate receptors (Verkhratsky and Kettenmann,
The major finding in the present study was the functional relationship between SOCE, STIM proteins and ionotropic AMPARs. We showed that interactions between endogenous STIM1/STIM2 and AMPAR GluA subunits occur in cortical neurons and that SOCE-dependent mechanisms affect AMPA-mediated [Ca2+]i rise. TG-induced SOCE was decreased 2.2–3.7 times in the presence of the potent competitive AMPAR antagonists CNQX and NBQX, suggesting that Ca2+ store depletion leads to Ca2+ influx by AMPARs. There exists a possibility that addition of calcium activates synapses, causing a large increase in network activity, and consequently an influx of calcium through NMDA and AMPARs. Indeed, Baba et al. (
STIM2 was recently proposed to shape basal transmission in excitatory neurons and is involved in the formation of dendritic spines and essential for the cAMP/PKA-dependent phosphorylation of AMPARs (Garcia-Alvarez et al.,
In the cerebral cortex of mammals majority of pyramidal cells (glutamatergic) contains GluA2 subunit of AMPAR, whereas non-pyramidal cells (mainly GABAergic inhibitory interneurons) preferentially express GluA1-specific mRNA (Martin et al.,
ML-9 is a myosin light chain kinase (MLCK) inhibitor that is commonly used to inhibit SOCE (Watanabe et al.,
The inhibitory effect of ML-9 on SOCE is not completely understood, but it seems to be unrelated to the inhibition of MLCK. Earlier work revealed that knocking down MLCK using siRNA or wortmannin (another MLCK inhibitor) does not affect SOCE or STIM1 redistribution (Smyth et al.,
It is well known that a sustained Ca2+ entry is linked to InsP3-dependent Ca2+ store depletion induced by activation of metabotropic glutamate receptors (Berridge,
In the present study, we found that SOCE in neurons was approximately two-times lower than in glial cells. This seems to reflect the larger size of Ca2+ stores in glia, in which TG-induced SOCE should lead to greater Ca2+ influx than in neurons. Similar observations were described previously (Arakawa et al.,
Recently, SOCE in neurons has become the subject of intense research with regard to disturbances in Ca2+ homeostasis that are observed in several neurological disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, chronic epilepsy, amyotrophic lateral sclerosis, painful nerve injury and cerebral ischemia (Leissring et al.,
In conclusion, the present data demonstrate that SOCE is functional in neurons as an AMPA-dependent Ca2+ signal. Together with the studies by Park et al. (
JG-B conceived, designed and performed experiments, analyzed the data, and wrote the manuscript. MS performed the experiments, analyzed the data, and wrote the manuscript. JK designed the experiments, analyzed the data and wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by funds from the National Science Centre (DEC-2011/01/D/NZ3/02051 to JG-B).