Abstract
STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca2+ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (ISOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. ISOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La3+. Overexpression of STIM1 or Orai1 alone did not induce any change in ISOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in ISOC. A large increase (>10-fold) in ISOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in ISOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to ISOC activation.
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Introduction
Ca2+ has long been recognized as an important second messenger, and intracellular Ca2+ has been shown to play an important role in regulating a variety of physiological processes in both excitable and non-excitable cells. Intracellular Ca2+ concentration ([Ca2+]i) may be increased through various mechanisms (1) the release of Ca2+ from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), (2) Ca2+ entry from extracellular space through voltage-operated Ca2+ channels (VOCCs) or receptor-operated channels (ROCs) or store-operated channels (SOCs) (Barritt 1999; Parekh and Putney 2005). The functional control of SOCs is dependent upon the Ca2+ concentration in the ER/SR lumen: when intracellular Ca2+ stores are full, Ca2+ influx does not occur; as the stores deplete, however, Ca2+ entry develops. The concept of SOCE or ISOC was proposed in 1986 (Putney 1986) and developed more fully during the past 20 years. With the introduction of electrophysiological techniques, several ISOC types have been characterized including the Ca2+ release-activated Ca2+ current (ICRAC), typically found in T lymphocytes and mast cells (Roos et al. 2005). The molecular compositions of SOCs, and the exact signaling pathways that activate these channels remain debated. In recent years, several independent groups have identified STIM1 and Orai1 to be essential components of SOCs, especially the CRAC channels (Parekh 2006; Huang et al. 2006; Prakriya et al. 2006).
STIM1 protein was reported to contain several important domains: an EF-hand domain, and a sterile-alpha motif (SAM) in the N-terminus, and a coiled coil region, and a lysine-rich region in the C-terminus. It is thought that STIM1 is the Ca2+ store-sensing component of SOCs and mutations in acidic residues within the Ca2+-binding pocket of the EF-hand domain of STIM1, which presumably lower the affinity for Ca2+, produce constitutive Ca2+ entry that is independent of store depletion (Smyth et al. 2006; Liou et al. 2005; Zhang et al. 2005).
Orai1 was first discovered from genetic linkage analysis performed to identify mutations contributing to a rare form of clinical severe combined immunodeficiency (SCID), and it was predicted to be a membrane-spanning protein with four transmembrane domains (Feske et al. 2006; Vig et al. 2006). The SCID patients with the mutation for Orai1 were reported to have a decreased ICRAC activity in T cells. This was confirmed by the failure of the mutant Orai1 to rescue the ICRAC activity when transfected into T cells and fibroblasts, whereas the wild-type Orai1 did (Feske et al. 2006).
There is also increasing evidence that members of the canonical subgroup of transient receptor potential channels constitute SOCs (Parekh and Putney 2005; Pedersen et al. 2005; Albert et al. 2007). Currently, TPRC1 is thought to be the most likely candidate for the formation of some SOCs. Several recent studies had shown that SOCs were impaired in different mammalian cells when TRPC1 expression was inhibited using antibodies, antisense or siRNA methods (Saleh et al. 2006, 2008; Takahashi et al. 2007).
As a type of non-excited cells, hepatocytes (liver cells) are functionally regulated by intracellular Ca2+. Despite the role as a second message (Barritt et al. 1981), changes in [Ca2+]i in hepatocytes constitute an essential intracellular signaling event which functions in unison with numerous other signaling pathways, including the extensive protein kinase network, in regulating hepatocyte and liver function under normal and pathological conditions (Gaspers and Thomas 2005; Teoh and Farrell 2003; Nieuwenhuijs et al. 2006). Furthermore, the functions of SOCs have been shown in hepatocytes and liver cell lines (Graf and Häussinger 1996; Auld et al. 2000). Also, in accord with investigations in other cell types, the results of recent experiments with liver cells indicate that STIM1, Orai1 and TRPC1 exist in rat liver cells and play essential roles in the formation and regulation of SOCs (Aromataris et al. 2008; Barritt et al. 2009; Chen and Barritt 2003; Brereton et al. 2001).
Although the characteristics of SOCs in animal hepatocytes have been reported (Graf and Häussinger 1996; Barritt et al. 2009; Chen and Barritt 2003), the study on SOCs in human liver cells is still lacking. Since STIM1, Orai1 and TRPC1 are all necessary for SOCs, the interactions among these proteins provide the mechanism for activating SOCs in different mammalian cells (Li et al. 2007; Jardin et al. 2008; Ong et al. 2007). However, there is no evidence for functional interaction among the three proteins in the activation of SOCs in human liver cells. The aim of the present study was to investigate the roles of STIM1, Orai1 and TRPC1 on the activation of SOCs in human liver cells.
Materials and methods
Reagents
Cell culture supplies: RPMI-1640, trypsin-EDTA, and antibiotics were purchased from Gibco. Fetal bovine serum (FBS) was bought from Hyclone. Thapsigargin (Tg), Fast Red TR and Naphthol AS-MX phosphate were products of Sigma-Aldrich. Fluo-4/AM, TRIZOL and Lipofectamine 2000 were purchased from Invitrogen. Antibodies for STIM1 and Orai1 were acquired from Proteintech, TRPC1 antibodies were bought from Abnova. Peroxidase-conjugated secondary antibodies were from Beijing Zhongshan Goldenbridge Company. 2-APB was from Calbiochem. All other reagents were of reagent grade.
Cell culture, plasmid construction and transfection
Human liver cell line, HL-7702, purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, was cultured at 37°C in 5% (v/v) CO2 in air in RPMI-1640 medium and 10% (v/v) heat-inactivated FBS. pBS-T-TRPC1 plasmid was constructed and verified as previously described (Rao et al. 2006). STIM1-mORANGE and Orai1-EGFP were gifts from Prof. Tao Xu (Institute of Biophysics, Chinese Academy of Sciences, China). HL-7702 cells were allowed to grow to ~80% confluence and transfected with required DNA at concentration of 1 μg/ml, using Lipofectamine 2000 and protocols supplied by the manufacturer.
RNA isolations, first strain cDNA synthesis, and semiquantitative RT-PCR analysis
Total RNA was extracted from HL-7702 cells using TRIZOL reagent according to the instructions offered by the manufacturer. The primer sequence used for TRPC1, STIM1 and Orai1 are given in Table 1. First strand cDNA synthesis and RT-PCR were performed as described previously (Rao et al. 2006). After PCR, 10 μl of the PCR product were analyzed on a 1% agarose gel. Band recognition and semiquantitative calculation were all performed by the software, Gelpro Analyzer 4.0 (Media Cybernetics, USA).
Western blotting
HL-7702 cells were cultured and transfected with the indicated plasmids as described earlier. The cells were harvested, lysed and stored at −80°C. An aliquot of 20 μg proteins was diluted to 45 μl in the loading buffer (44.6 mM Tris at pH 6.8, 1.6 mM EDTA, 44.5 mM boric acid, 10% glycerol, 2% SDS, 5% β-mercaptoethanol and 0.1% bromphenol blue), separated by 3 and 9% SDS–PAGE gel, transferred to polyvinylidene difluoride membranes, and probed with anti-TRPC1, anti-STIM1 and anti-Orai1 antibodies. Peroxidase-conjugated secondary antibodies were used to label the proteins. The proteins on the membranes were stained by a staining solution containing Fast Red TR and Naphthol AS-MX phosphate. Finally, the red protein bands were cut and placed in 200 μl of dimethyl sulfoxide (DMSO) for discolouration, and the absorbance of the resulting DMSO solution was measured in a micro plate reader (Thermo, USA) at 492 nm.
Electrophysiology
Whole-cell patch clamping was performed at room temperature (22–25°C) using a computer-based patch-clamp amplifier (EPC-10, HEKA Electronics, Lambrecht/Pfalz, Germany) and PatchMaster software (HEKA Electronics). HL-7702 cells grown in 35-mm dishes were perfused with a standard external solution containing the following (in mM): 140 NaCl, 4 CsCl, 2 MgCl2, 10 CaCl2, 10 D-glucose and 10 Hepes (pH 7.4, adjusted with NaOH). The patch pipettes were pulled from borosilicate glass and fire polished. Pipette resistance ranged between 4 and 8 MΩ after filling with the standard intracellular solution containing (in mM): 15 CsCl, 135 Cs-glutamate, 10 Hepes and 10 EGTA (pH 7.2, adjusted with CsOH). In all whole cell experiments, the recording started when the series resistance dropped to below 20 MΩ. After establishment of the whole cell configuration, voltage ramps of 100 ms duration, spanning a range of −160 to +100 mV, were delivered from a holding potential of 0 mV every 2 s over a period of 300–800 s. All voltages were corrected for a liquid-junction potential of 17 mV (estimated by JPCalc). Capacitative currents were determined and compensated for automatically by the EPC-10 amplifier. The maximal currents at −100 mV were used for statistical analysis.
Calcium imaging
For imaging experiments, HL-7702 cells and transfected cells were seeded in glass-bottomed microwell dishes the day prior to experimentation. Then cells were loaded with 6.7 μmol/L Fluo-4/AM in the standard external solution at 37°C for 30 min, followed by three washes in PBS and 15 min incubation in cultural medium to allow de-esterification of the dye. Changes of [Ca2+]i in cells were monitored at 5 s intervals with a Leica TCS SP5 confocal laser scanning microscope equipped with an argon laser (Leica, Wetzlar, Germany). The 495 nm laser was used for Fluo-4 excitation, and emitted fluorescence was collected through a 500–530 nm window of the detector. LAS AF was applied for hardware control, image acquisition and image analysis.
Single cell RT-PCR
Single cell RT-PCR was performed to determine the cells measured by patch clamp were successfully transfected with the desired cDNA using the previously described method with modification (Rabe et al. 1998). In this experiment, harvesting cytoplasm of patched cells was performed by a gentle suction applied to the pipette. The tip of the pipette was then broken in a PCR tube containing the required reagents for reverse transcription, which was followed by PCR using specific primers described in Table 1. The first PCR amplification was performed as follows: a pre-denaturation at 94°C for 3 min, followed by 25 (for STIM1 and Orai1) or 30 (for TRPC1) cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and synthesis at 72°C for 60 s, and then a last extension step at 72°C for 5 min. The second round of PCR was then performed using 5 μl of the products of the first amplification as templates. Each gene fragment was amplified individually using its specific primer pair by performing 35 cycles for TRPC1 and 30 cycles for STIM1 and Orai1 (as described above). PCR products were analyzed using agarose gel electrophoresis.
Statistical analysis
Unless specially noted, all current traces were corrected for leak currents. Data analysis was conducted using IGOR Pro 5.01 (Wavemetrics, Portland, OR, USA). Means standard deviation (SD) were calculated according to standard procedures. Statistical significance of difference between test samples and controls was determined by using the DUNNET t test. Asterisks denote statistically significant difference with p values less than 0.05 (*) and 0.01 (**).
Results
Native store-operated Ca2+ currents in HL-7702 cells
It has been shown that ISOC is present in rat liver cells and human hepatomas (Rychkov et al. 2001; Jiang et al. 2001; El Boustany et al. 2008). In this study, current response to Ca2+ store depletion induced by Tg was tested in wild type HL-7702 cells by whole-cell patch clamp recording. As shown in Fig. 1a, the store-depletion activated Ca2+ currents have a linear current–voltage relationship at negative potentials and a slight outward rectification at positive potentials, with a reversal potential at near 0 mV. The peak current density was 0.62 ± 0.07 pA/pF at −100 mV (n = 6), and it was reduced to 0.29 ± 0.04 pA/pF and 0.29 ± 0.02 pA/pF by La3+ (100 μM) and 2-APB (50 μM), respectively (Fig. 1b).
STIM1, Orai1 and TRPC1 expression in HL-7702 cells
To determine if STIM1, Orai1 and TRPC1 are expressed in the HL-7702 cells, we first used RT-PCR. Using gene-specific primers described in Table 1, the corresponding products of STIM1 (343 bps), Orai1 (342 bps) and TRPC1 (455 bps), were amplified, confirming the expression of these three genes in HL-7702 cells. Second, we tested the expressions of the three proteins by western-blot analysis using specific antibodies. The results showing molecular mass of 77 kDa for STIM1, 35 kDa for Orai1 and 88 kDa for TRPC1 are in agreement with the expected sizes of these proteins, demonstrating that all three proteins are constitutively expressed in HL-7702 cells (Fig. 2a, c, e–h).
STIM1 and Orai1 enhance SOC functions in HL-7702 cells
Co-overexpression of STIM1 and Orai1 reconstituted ICRAC in several different cell types, including HEK293, lymphocytes, mast cells, etc. (Barritt et al. 2009; Zhang et al. 2006; Soboloff et al. 2006; Peinelt et al. 2006). However, overexpressing either STIM1 or Orai1 failed to increase ICRAC. We observed similar phenomena in HL-7702 cells. Overexpression of STIM1 or Orai1 alone did not induce any change in ISOC (the peak current density of STIM1-transfected cells was 0.64 ± 0.06 pA/pF and it remained as 0.60 ± 0.08 pA/pF when Orai1 was overexpressed in HL-7702 cells), while co-expression of STIM1-mORANGE and Orai1-EGFP together in HL-7702 cells, as shown by RT-PCR and western blotting (Fig. 2a–d) as well as images taken for the same HL-7702 cell by confocal laser microscopy (Fig. 3a), resulted in an increase in the Tg-evoked ISOC with obvious inward rectification at negative potentials, reminiscent of that of ICRAC (Fig. 3b, c). However, there is also an obvious increase in the outward current and the reversal potential was again near 0, suggesting that the newly formed channels due to STIM1 and Orai1 co-expression in the liver cells are different from the CRAC channel, in terms of ion selectivity. In calcium imaging experiments, we show that overexpressing STIM1 + Orai1 caused an increase in SOCE (Fig. 3d, e).
Significant role of TRPC1 on the activation of SOCs in HL-7702 cells
Unlike STIM1 and Orai1, transfection of TRPC1 alone substantially increased ISOC. As shown in Fig. 4a, an approximate 2.5-fold increase in peak current density of ISOC was caused by TRPC1 overexpression. Single cell RT-PCR experiments were performed to confirm that the expression levels of TRPC1 were elevated by transfection in the patched cells. Figure 4b shows the images of a cell after electrophysiological recording before (left) and after (right) a suction was applied to extract the cell content. The shrinkage of cell membrane was recognized as successful harvesting of cell cytoplasm.
Compared to TRPC1 overexpression alone, co-overexpression of STIM1 and TRPC1 induced a further increase in ISOC. The time course in current development at −100 mV in Fig. 4c and I–V curves in Fig. 4d show representative traces of ISOC developed in cells co-transfected with TRPC1 and STIM1. An approximate 10-fold increase in ISOC was obtained by the co-expression of TRPC1 + STIM1. Notably, no further increase in ISOC was detected with the addition of Orai1. In cells that co-expressed STIM1, Orai1 and TRPC1, the time course of ISOC development and the shape of the I–V curves at the peak current are not different from those that co-expressed STIM1 and TRPC1 (Fig. 4f, g). The peak current density of the triple expressing cells is also similar to cells that expressed STIM1 and TRPC1 (Fig. 4h; Table 2). For all cells, we also carried out single cell RT-PCR experiments after the electrophysiological recording to confirm that mRNAs for the three proteins were indeed overexpressed in the patched cells (Fig. 2h). Peak current densities at −100 mV for individual cells are listed in Table 2.
Discussion
The present study provides the first direct evidence that functional interactions among STIM1, Orai1 and TRPC1 activate SOCs in HL-7702 cells. This was indicated by the inhibitory effects of SOC inhibitors and the enhancement of ISOC in cells that overexpressed the three proteins. With a near zero reversal potential, ISOC recorded in this study is not identical to the traditional ICRAC. Furthermore, co-overexpression of the STIM1 and Orai1 proteins resulted in an increase in ISOC not only at negative but also positive potentials. It is possible that the outward currents at the positive potentials represent activation of Ca2+-dependent Cl− channels, which had been found in rat liver cells (Aromataris et al. 2006).
It was reported that the overexpression of STIM1 alone caused a zero-to-modest increase of ICRAC in HEK293 and Jurkat cells, whereas Orai1 overexpression alone did not affect ICRAC induced by store depletion in HEK293 cells, and instead, caused a small reduction in ICRAC in Jurkat cells (Roos et al. 2005; Cheng et al. 2008; Spassova et al. 2006). Our findings in HL-7702 cells are consistent with these previous results, indicating that STIM1 or Orai1 alone was either insufficient to generate large ISOC or they are stoichiometrically linked limiting each others’ ability to generate an increased ISOC (Peinelt et al. 2006). Among all TRP channels, evidence for TRPC1 to form SOCs is perhaps the strongest. It has been shown that functional expression of human TRPC1 resulted in a modest increase in Tg-evoked Ca2+ influx in COS cells (Zhu et al. 1996) and HEK293 cells (Cheng et al. 2008). In the present study, the experimental results also show association of TRPC1 overexpression with an increase of ISOC in HL-7702 cells. This supports the view that TRPC1 is involved in the formation of SOCs. A possible mechanism to account for this (as shown in Fig. 5) is that SOCs in the plasma membrane consist of TRPC1 and other regulatory subunits (including Orai1); store depletion causes STIM1 aggregation and binding to either TRPC1 or a regulatory subunit, resulting in conformation change and channel opening.
There is also increasing evidence in several different cell types that co-overexpression of two proteins (STIM1 + Orai1 or STIM1 + TRPC1) or three proteins (STIM1 + Orai1 + TRPC1) leads to enormous ISOC, which is significantly larger than the corresponding native currents evoked using the same experimental protocol (Ong et al. 2007; Peinelt et al. 2006; Cheng et al. 2008). This suggests that either Orai1 or TRPC1 may interact with STIM1 and respond to store depletion signal, and each may be sufficient to control the magnitude of ISOC, although the coexistence of the three proteins is not detrimental to activate SOCE. This hypothesis is, however, distorted by the inconsistent data in the existing literature where the enhancement of ISOC caused by co-overexpression is not fully equal in different experiments, even in the same type of cells (Li et al. 2007; Cheng et al. 2008). Presumably, variable transfection efficiencies may partly be responsible for the differences in the degree of ISOC increase. This argues for the importance of maintaining the appropriate stoichiometry of all key proteins and factors in the regulation of SOCs.
Taken together, STIM1, Orai1 and TRPC1 are all important in activating ISOC and it can infer from our experimental findings that overexpression of STIM1 or Orai1 alone will not increase ISOC while overexpression of TRPC1 will, co-overexpression of STIM1 + Orai1, STIM1 + TRPC1 or STIM1 + Orai1 + TRPC1 can all dramatically increase ISOC. Here we assume at least three distinct possible pathways of SOC activation in HL-7702 cells. The first pathway proposes the existence of a protein channel consisting of Orai1 and TRPC1, and although it is kept inactive at rest, once Ca2+ store is depleted, it is detected by the Ca2+ sensor in the ER and STIM1 will then couple to Orai1, promoting a change in conformation of SOCs to open the channels. The other two pathways are more complex. They support the channel is consisted of TRPC1 and another regulatory subunit (may be TRPC4, TRPC5 or other proteins), either STIM1 or other mediators (IP3, ATP or cGMP, etc.) may interact with the subunit and activate SOCE. Thus, opening of Orai1-based SOCs highly depend on STIM1, whereas STIM1 is only one of the activators in the latter two pathways.
In conclusion, STIM1, Orai1 and TRPC1, the three proteins that regulate normal functions and pathological processes in liver cells since Ca2+ acts as an important second messenger. In wild type cells, variant Ca2+ channels constitute an equilibrious system and maintain physiological state. Under pathological conditions, the expression of the three proteins may change and thereby impairs the intracellular Ca2+ concentration regulation and hence the functions of hepatocytes. Thus, STIM1, Orai1 and TRPC1 may be new targets for disease prevention or therapy of the ion channel disorders or channelopathies of liver in clinic.
Abbreviations
- 2-APB:
-
2-Aminoethoxydiphenyl borate
- EDTA:
-
Ethylene diamine tetraacetic acid
- EGFP:
-
Enhanced green fluorescence protein
- EGTA:
-
Ethylene glycol tetraacetic acid
- ER:
-
Endoplasmic reticulum
- FBS:
-
Fetal bovine serum
- ICRAC :
-
Ca2+ release-activated Ca2+ current
- ISOC :
-
Store-operated current
- PBS:
-
Phosphate-buffered saline
- ROCs:
-
Receptor-operated channels
- RT-PCR:
-
Reverse transcription-polymerase chain reaction
- SCID:
-
Severe combined immunodeficiency
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SOCE:
-
Store-operated Ca2+ entry
- SOCs:
-
Store-operated channels
- SR:
-
Sarcoplasmic reticulum
- STIM1:
-
Stromal interacting molecular 1
- Tg:
-
Thapsigargin
- TRPC1:
-
Transient receptor potential canonical 1
- VOCCs:
-
Voltage-operated Ca2+ channels
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Acknowledgments
We specially thank Prof. Tao Xu (Institute of Biophysics, Chinese Academy of Sciences, China) for kindly providing STIM1-mORANGE and Orai1-EGFP plasmids. We thank Prof. Michael Xi Zhu (Department of Neuroscience and Center for Molecular Neurobiology, Ohio State University, USA), Dr. Lyn Trapuzzano and Dr. Changqing Xu (Blanchette Rockefeller Neurosciences Institute, West Virginia University, USA), for critical reading of the manuscript. This work was financially supported by the National Science Foundation of China (Grants 30270532 and 30670744) and Tsinghua-Yue-Yuen Medical Science Foundation (No. 20240000531 and No. 20240000547) to ZM Zhang.
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Zhang, ZY., Pan, LJ. & Zhang, ZM. Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells. Amino Acids 39, 195–204 (2010). https://doi.org/10.1007/s00726-009-0398-5
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DOI: https://doi.org/10.1007/s00726-009-0398-5