Functional significance of human trp1 and trp3 in store-operated Ca2+ entry in HEK-293 cells

Xiaoyan Wu, György Babnigg, and Mitchel L. Villereal

Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Drosophila trp (transient receptor potential) gene appears to encode the Drosophila store-operated channel (SOC), and some mammalian trp homologues have been proposed to encode mammalian SOCs. This study provides evidence for the expression of three trp homologues (Mtrp2, Mtrp3, and Mtrp4) in fibroblasts from wild-type and src knockout mice, and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in human embryonic kidney (HEK-293) cells based on RT-PCR techniques. In HEK-293 cells stably transfected with a 323-bp Htrp3 antisense construct (Htrp3AS), Northern blot analysis revealed that the expression of a 4-kb transcript was dramatically suppressed in comparison to that observed in cells stably transfected with a short Htrp3 sense construct (Htrp3S). Activity of SOCs, monitored as Ba2+ influx following Ca2+ store depletion with thapsigargin, was reduced by 32% in Htrp3AS cells in comparison with Htrp3S cells. Transient transfection of a 369-bp Htrp1 antisense construct in cells stably expressing Htrp3AS induced a higher level of inhibition (55%) of store-operated Ca2+ entry. These data suggest that Htrp1 and Htrp3 may be functional subunits of SOCs.

human transient receptor potential proteins; transient receptor potential; antisense cDNA; calcium store depletion; thapsigargin; Ba2+ influx; stable transfection; human embryonic kidney cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTRACELLULAR CALCIUM ([Ca2+]i) is crucial for countless cellular functions and processes (3, 4, 50). In excitable cells such as neurons, muscle, and endocrine cells, the level of [Ca2+]i is regulated by well characterized voltage-operated Ca2+ channels, i.e., L-, T-, N-, P- and Q-type channels (47). However, in nonexcitable cells such as hepatocytes, T lymphocytes, fibroblasts, vascular endothelial cells, and epithelial cells in the respiratory and digestive tracts, much of the Ca2+ entry is carried by voltage-insensitive Ca2+ channels which are less well characterized (11, 13). A significant portion of Ca2+ influx into nonexcitable cells is mediated by store-operated Ca2+ channels (SOCs) activated by emptying of the intracellular inositol trisphosphate (InsP3)-sensitive Ca2+ stores (10, 40). Activation of SOCs represents a mechanism that serves both to maintain an elevated cytosolic Ca2+ while InsP3 levels remain high and to replenish depleted intracellular storage compartments once the InsP3 levels decline. Store depletion has been reported to generate a variety of different Ca2+ currents (34). The first such current described, which is via the best characterized and most Ca2+ selective SOC, is called the Ca2+-release-activated Ca2+ current (Icrac) (20, 21). Single-channel conductance of Icrac was found to be quite small (26, 27, 35). Although it is clear that Icrac behaves as a SOC, it may only represent one subtype of SOCs, because there is a large variation in characteristics observed for SOCs measured in different tissues (24, 41). Although the concept of SOCs has been well documented by both fura 2 and electrophysiological data, the molecular identity of these channels and their mode of regulation have not been clearly identified.

Regarding the regulation of store-operated Ca2+ entry (SOCE), our previous studies in cultured fibroblasts show that bradykinin and thapsigargin stimulate a SOCE that can be, in both cases, blocked by tyrosine kinase inhibitors such as genistein and tyrphostin (7, 25). In addition, Ca2+ influx following store depletion is dramatically diminished in Src- fibroblasts, an src-/src- cell line derived from src knockout mice. The level of SOCE can be restored to control levels by transfection of Src- cells with chicken src (1). These findings strongly suggest that the tyrosine kinase src plays a role in the regulation of SOCE. However, the question of which protein(s) serves as the target of the src tyrosine kinase activity in this process remains to be investigated and must await the clarification of which protein mediates SOCE. We undertook the present study in human embryonic kidney (HEK-293) cells as a first step in identifying which protein may serve as the SOC, and therefore may serve as a substrate for src kinase activity.

Trp (transient receptor potential), a Drosophila gene required in phototransduction (18, 29), encodes the best characterized protein mediating SOCE (16, 36, 44, 48). Multiple mammalian trp homologues have been identified; the sequence of cDNAs for trp1 (51, 53), trp3 (51), trp4 (55), and trp6 (12, 19) from human and trp 1-6 from mouse (36, 55) have been reported. Some of the trp homologues appear to have splice variants (14, 42). At the molecular level, the trp homologues share a common structure, i.e., a core of six transmembrane domains along with ankyrin repeats at the NH2 terminus of the protein (5). Based on the sequence similarity, the list of trp family members is expanding rapidly. Three other mammalian trp homologues were reported recently. One is the mouse melastatin gene (Mlsn1) that may play a role in suppression of cancer metastasis (22). A second is the capsaicin receptor, a heat-activated ion channel that functions in the pain-transduction pathway (8). A third is HtrpC7, which is highly expressed in brain (31). Among the trp homologues, functional expression of full-length cDNAs encoding human trp1 or trp3 in COS-M6 cells leads to a significant enhancement of thapsigargin-induced Ca2+ influx as measured by fura 2 (55). Likewise, heterologous expression of trp homologues in other studies also resulted in enhanced SOCE or in enhanced current in response to store depletion (17, 23, 28, 37-39, 46, 49, 57). Furthermore, expression of small portions of six mouse trp constructs in Ltk- fibroblast cells, all in antisense orientation, eliminates carbachol-induced Ca2+ influx (55), suggesting that one or more of these trp homologues is essential for SOCE. However, there also have been a number of recent publications that suggest that expression of trp homologues results in channel activity which is not dependent on store depletion (6, 33, 43, 54). These inconsistencies could result from differences in background in the cells used for trp expression since recent studies suggest that channel characteristics can be influenced by coexpression of multiple trp family members (16).

To avoid these problems inherent to expression studies, we used the antisense cDNA approach to investigate which trp proteins are involved in SOCE in HEK-293 cells. In the present study, we identified the endogenous trp homologues expressed in mouse fibroblasts and human embryonic kidney fibroblasts (HEK-293 cells). We then developed and expressed partial antisense constructs in HEK-293 cell lines. We present evidence that both human trp3 and human trp1 are important mediators of SOCE.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Materials. Fura 2 free acid, fura 2-AM, and Pluronic F-127 were purchased from Molecular Probes; thapsigargin from LC Laboratory; Nusieve GTG agarose from FMC BioProducts; G418 from Mediatech; Hanks' balanced salt solution (HBSS), Ca2+-free, Mg2+-free, HCO-3-free HBSS, and DMEM from Life Technologies GIBCO BRL; and [alpha -32P]dCTP from Amersham. All other chemicals were purchased from Sigma.

Isolation of cDNA encoding fragments of trps by RT-PCR. The poly(A)-RNA was isolated from three different cell lines, src+ mouse fibroblasts, src- mouse fibroblasts, and HEK-293 cells, using guanidinium thiocyanate extraction followed by an oligo(dT) binding method (QuickPrep Micro mRNA Purification Kit; Amersham Pharmacia Biotech). The first strand cDNA was reverse transcribed using an oligo(dT) primer, and it was then amplified directly using PCR (SuperScript Preamplification System; Life Technologies GIBCO BRL). Four pairs of primers were designed based on human gene sequence (Htrp), and six pairs of primers based on mouse gene sequence (Mtrp) were obtained from the GenBank database: Htrp1-U31110; Htrp3-U47050; Htrp4 (personal communication from Drs. X.-Z. S. Xu and C. Montell, Johns Hopkins School of Medicine); Htrp6-AF080394; Mtrp1-U40980; Mtrp2-U40981; Mtrp3-U40982; Mtrp4-X90697; Mtrp5-U40984; and Mtrp6-U49069. Sequence similarity analysis was performed using software by Genetics Computer Group. The sequences of the oligonucleotide primers as well as exact coding location are described in Table 1.

                              
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Table 1.   Sequences of primers used for RT-PCR reactions

The hot-start PCR was performed with Taq polymerase for 30 cycles: the initial denaturation was at 94°C for 5 min, which was followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 30 s. A final extension was performed at 72°C for 3 min. The PCR conditions were optimized to 30 cycles annealing at 55°C for Htrp1 and Htrp3 homologues, to 35 cycles annealing at 53°C to detect Htrp4 and Htrp6 homologues. The PCR reaction mixture (48 µl) consisted of ~22% of first strand cDNA as a template, 100 picomoles of each primer, 5 µl of 10× PCR buffer (200 mM Tris · HCl, pH 8.4, 500 mM KCl), 5 µl of 25 mM MgCl2, 1 µl of 10 mM dNTP mix, and 2.5 units of Taq DNA polymerase (Life Technologies GIBCO BRL). Two positive controls, one a commercial primer for control mRNA and the other a beta -actin primer for mRNA from mouse cells or HEK-293 cells, and a negative control without RT were performed alongside all experimental samples.

The cDNA fragments of Htrp1 (369 bp) and Htrp3 (323 bp) from RT-PCR reactions were separated by electrophoresis in a 3% GTG-agarose gel. The corresponding bands were cut out of gels, extracted (QIAEX II Gel Extraction 150), and subcloned into an eukaryotic TA cloning vector pCR3.1 that accepts products in both the forward and reverse directions (eukaryotic TA cloning Kit-Bidirectional; Invitrogen). The clones were selected randomly, and the cDNAs were purified (QIAGEN Plasmid Maxi Kit 25) and sequenced. The antisense constructs encoding Htrp1 and Htrp3 were expressed in HEK-239 cells, whereas the corresponding sense constructs were expressed for the controls.

Cell culture. Both mouse fibroblasts and HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine. Cells were grown in an incubator at 37°C with humidified 5% CO2-95% air.

Stable transfection of antisense Htrp3 construct. HEK-293 cells were transfected with the Htrp3 antisense construct (Htrp3AS cells) using the Ca2+ phosphate method. HEK-293 cells transfected with an Htrp3S construct (Htrp3S cells) were used as control. G418-resistant transformants were selected using 500 µg/ml G418. All of the surviving clones (~200) were pooled together to generate cell lines stably expressing either Htrp3AS or Htrp3S constructs. Cells up to passage 30 were used for Northern blot analysis as well as for Ca2+ imaging.

Transient expression of Htrp1 antisense constructs in Htrp3AS cells. Htrp3AS cells or Htrp3S cells were plated onto 100-mm dishes 6 h before transfection. They were then transfected with the Htrp1 antisense construct or the Htrp1 sense construct, respectively. The Ca2+ phosphate method was used for transient transfection, and its efficiency was between 40-60% as determined by the fluorescence of cells transfected in parallel with a construct for the green fluorescence protein. After 24 h of transfection, cells were harvested and plated onto coverslips to be used 24 h later for fura 2 Ca2+ imaging.

Northern blot analysis. Poly(A)-RNA was extracted from cells stably expressing Htrp3AS or Htrp3S constructs, resolved in a 1% agarose gel, and transferred to a Hybond-N nylon membrane by an electrophoretic method for 2 h at room temperature. Transfer solution consisted of 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA (pH 8.4). The Northern blot was prehybridized for 2 h at 45°C in XOTCH solution (10 mM EDTA, 100 mM NaH2PO4, 7% SDS, 1% BSA, 15% formamide) and then hybridized for 20 h at 45°C in the same buffer containing 32P-labeled probe (8 × 106 cpm/ml). After washing with 2× SSC/0.05% SDS (20× SSC: 3 M sodium chloride, 300 mM sodium citrate, pH 7.0) for 20 min at 45°C, the blot was exposed to X-ray film at either room temperature or -70°C for desired periods of time. The probe for Htrp3 was made from the fragment of the Htrp3 cDNA and a control probe was a human cDNA for glyceraldehyde-3-phosphate dehydrogenase. Both probes were labeled with [alpha -32P]dCTP (DECAprime II DNA labeling kit; Ambion).

Ca2+ imaging. [Ca2+]i concentration was measured in individual cells using the fluorescent indicator fura 2 (7). Transfected cells were plated onto 25-mm coverslips one day before the experiment. On the experimental day, cells were washed twice with a HEPES-buffered HBSS (HHBSS), and loaded with 5 µM fura 2-AM that was dissolved in HHBSS supplemented with 1 mg/ml BSA, 0.025% Pluronic F127 for 30 min, and then unloaded in HHBSS for another 30 min. The coverslip was mounted onto a chamber that was placed on the stage of a Nikon inverted epifluorescence microscope. The cells were excited alternatively at 340- and 380 nm. The image was captured by an SIT camera and transmitted to a computer. The captured 340- and 380-nm images were ratioed pixel by pixel. The [Ca2+]i value for each cell was established from a calibration curve based on fura 2 potassium salt. The average response of ~800 cells from each coverslip is represented as one trace. Cells inside the chamber were perfused by an eight-channel syringe system. Nominally Ca2+-free HBSS was prepared by treating Ca2+-free, Mg2+-free, HCO-3-free HBSS with Chelex-100, then adding MgCl2 to a final concentration of 1 mM. A measure of SOCE was obtained by subtracting the slope of the Ba2+ leak (Ba2+ entry before store depletion) from the slope of Ba2+ entry after store depletion. For the data in Fig. 3, the mean Ba2+ leak flux of the sample population was used to correct the mean Ba2+ influx following store depletion, whereas for the data in Fig. 4, the correction was done on individual coverslips.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Endogenous expression of trp homologues. It has been reported that the endogenous message for SOCs in animal cells may be expressed at relatively low levels, which constitutes one of the major problems in detecting channel activity across the plasma membrane (2). Also, it has been known for some time that RT-PCR reactions can be influenced by a variety of factors, such as magnesium concentration, annealing temperature, and PCR cycles. Failure to optimize reaction conditions could lead to nonspecific signals or to the failure to see bands for expressed products (32). Therefore, our initial efforts were to optimize the conditions for each individual PCR product. As shown in Fig. 1A, 2.5 mM MgCl2 gave rise to a clear, single band, and it was then used in all RT-PCR reactions that we reported in this paper. We also tested annealing temperature and PCR cycles. We found that amplification for 30-35 cycles, along with a variety of annealing temperatures (53°C, 55°C, or 60°C), increases the chances of detecting low levels of expression and enables us to observe most isoforms of trp in a given cell line (data not shown).


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Fig. 1.   RT-PCR for detecting endogenous expression of transient receptor potential (trp) homologues in mouse Src- and Src+ fibroblasts, and human embryonic kidney (HEK-293) cells. Poly(A)-RNA was extracted from cells and first strand cDNAs were reverse transcribed. Hot-start PCR was performed with Taq polymerase for 30 or 35 cycles. Two positive controls (using a commercial primer for control mRNA and a beta -actin primer for mRNA from corresponding cell lines) and one negative control without RT were performed alongside all experimental samples. A: Mg2+ dependence of PCR used for detection of endogenous Htrp3 in HEK-293 cells. B: endogenous expression of mouse trp homologues in Src- fibroblasts. C: endogenous expression of mouse trp homologues in Src+ fibroblasts. D: endogenous expression of human trp homologues in HEK-293 cells.

We then used the optimized RT-PCR conditions to establish the distribution of trp in three mammalian cell lines: fibroblasts from wild-type mice (Src+ cells), fibroblasts from src knockout mice (Src- cells), and HEK-293 cells. As shown in Fig. 1, expression of Mtrp2, Mtrp3, and Mtrp4 was detected in both Src- (Fig. 1B) and Src+ (Fig. 1C) cell lines. Based on repeated experiments involving amplification at varying annealing temperatures and evaluation of transcript levels on agarose gels by ethidium bromide staining, the Mtrp3 band was always more intense in Src- cells, suggesting higher levels of Mtrp3 than in Src+ cells, whereas the Mtrp2 band was more intense in Src+ cells, suggesting a higher level of expression of Mtrp2 than in Src- cells. The intensity of the Mtrp4 band tended to be approximately the same in both Src- and Src+ cells. Mtrp1, Mtrp5, and Mtrp6 were not detected in either cell line.

As shown in Fig. 1D, in HEK-293 cells, Htrp1 and Htrp3 gave very strong bands following 30 PCR cycles. The Htrp4 and Htrp6 bands were detected following a total of 35 cycles of PCR. The trp2 and trp5 homologues were not detectable in HEK-293 cells even after 35 cycles at varying annealing temperatures.

Expression of Htrp3 mRNA in HEK-293 cells stably transfected with either Htrp3AS or Htrp3S constructs. To determine the role of Htrp3 in mediating SOCE, we made an Htrp3AS cDNA construct that we stably transfected into HEK-293 cells. Instead of selecting one or several clonal populations of transfected cells to study, we mixed the surviving clones (~200) together to make a heterogeneous population of cells expressing the Htrp3AS construct. We did this to assure that the large cell-to-cell variations of SOCE seen in the parent HEK-293 population did not impact the outcome of these experiments (2a).

mRNA was extracted from the HEK-293 cells stably expressing either Htrp3AS or Htrp3S constructs. The levels of Htrp3 mRNA were detected by Northern blot analysis using a 323 bp fragment of the Htrp3 cDNA as a probe. As shown in Fig. 2, the Htrp3 probe hybridized with a transcript of 4 kb in HEK-293 cells stably transfected with the Htrp3S construct. However, very little Htrp3 transcript was detected in HEK-293 cells stably transfected with the Htrp3AS construct.


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Fig. 2.   Expression of Htrp3 mRNA in HEK-293 cells stably transfected with Htrp sense (Htrp3S) or Htrp antisense (Htrp3AS) cDNA constructs. HEK-293 cells were stably transfected with Htrp3S or Htrp3AS constructs by the Ca2+ phosphate method. An initial population of G418-resistant transformants (~200 clones) was expanded. Poly(A)-RNA was extracted, resolved in 1% agarose gel, and transferred to a Hybond-N nylon membrane by electrophoretic method. Northern blot hybridized with a probe made from cDNA fragment of Htrp3 (upper panel). Result of same blot hybridized with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (lower panel). Blot is representative of three experiments.

Suppression of SOCE in HEK-293 cells stably expressing a Htrp3AS construct. To investigate the functional role of Htrp3 in mediating SOCE in HEK-293 cells, we compared SOCE in cells expressing the Htrp3AS construct to SOCE in cells expressing the Htrp3S construct (Htrp3AS vs. Htrp3S). We monitored the level of Ba2+ entry, via fura 2 image analysis, as a measure of the activity of Ca2+ entry pathways. Because Ba2+ is not pumped by Ca2+-ATPases, the level of Ba2+ entry is not complicated by a compensating pump activity as seen for Ca2+. We monitored both the Ba2+ leak (i.e., the slope of Ba2+ entry in the absence of store depletion) and the Ba2+ entry following store depletion by thapsigargin, a Ca2+-ATPase inhibitor that can deplete internal Ca2+ stores without a concomitant rise in InsP3 (45). Addition of thapsigargin in a Ca2+-free medium results in an initial transient Ca2+ peak which reflects the depletion of intracellular stores (25) and the removal of this Ca2+ from the cell by the plasma membrane Ca2+-ATPase. The Ba2+ leak, the transient Ca2+ release by thapsigargin, and the thapsigargin-stimulated Ba2+ entry are shown for a single representative coverslip where the response is averaged over ~800 cells in the microscope field (Fig. 3A). When one compares the transient Ca2+ peak of the curves for two representative coverslips (Htrp3AS cells vs. Htrp3S cells), there is little difference (Fig. 3B). A statistical analysis of the areas underneath the transient Ca2+ peaks, measured on all coverslips of each group, confirms that there is not a statistically significant difference between the average amount of Ca2+ released by thapsigargin in the Htrp3S and Htrp3AS groups (Table 2). These results suggest that the suppression of Htrp3 expression does not significantly alter the storage of Ca2+ or its release from its internal stores.


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Fig. 3.   Suppression of store-operated Ca2+ entry (SOCE) in HEK-293 cells stably transfected with an Htrp3AS construct. A: a comparison of basal leak of Ba2+ measured in a Ca2+-free HEPES-buffered Hanks' balanced salt solution (HHBSS) before depletion of Ca2+ stores and the Ba2+ influx following depletion of Ca2+ stores by thapsigargin. Data shown are for single representative coverslips with response averaged over ~800 cells. In cells in which the Htrp3AS construct was expressed, basal Ba2+ leak was slightly higher than in cells expressing the Htrp3S construct [Htrp3S (n = 19): 0.12 ± 0.01; Htrp3AS (n = 17): 0.17 ± 0.02]. B: thapsigargin-induced Ca2+ release in Htrp3S cells (fine line) or Htrp3AS cells (thick line) loaded with fura 2. Cells were perfused with HHBSS (0-20 s) followed by Ca2+-free HHBSS (21-800 s) during which time thapsigargin (1 µM) was added into chamber. Each trace represents average response of ~800 cells on a single coverslip. C: SOCE was determined by subtracting Ba2+ leak flux from Ba2+ influx following depletion of Ca2+ stores by thapsigargin. Curves shown are for SOCE averaged over 36 coverslips for Htrp3S cells and 32 coverslips for Htrp3AS cells. As indicated, SOCE in cells expressing trp3 sense construct (fine line) was higher than that in cells expressing trp3 antisense construct (thick line). D: statistical analysis of SOCE in Htrp3S cells in comparison with Htrp3AS cells. A value for SOCE was obtained for each individual coverslip and then the mean SOCE of the population determined for each cell type. Mean value of SOCE determined in Htrp3S cells is plotted as 100%. Htrp3 knockout resulted in a 32% inhibition of SOCE (Htrp3S: 0.56 ± 0.046 vs. Htrp3AS: 0.38 ± 0.021). * Statistically significant difference from Htrp3S cells (P < 0.0001). [Ca2+]i, intracellular Ca2+.


                              
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Table 2.   Statistical analysis of thapsigargin-induced Ca2+ peak from HEK-293 cells stably transfected with Htrp3 sense (Htrp3S) or Htrp3 antisense (Htrp 3AS) constructs

After Ca2+ store depletion and the return of [Ca2+]i to baseline, SOCE was assessed by the addition of a Ba2+- containing medium. After depletion of the stores, the Ba2+ influx was dramatically increased in comparison with the basal Ba2+ leak measured before store depletion (Fig. 3A). Such a Ba2+ leak occurred in both Htrp3S and Htrp3AS cell lines. Furthermore, cells expressing Htrp3AS construct had a slightly greater basal Ba2+ leak compared with cells expressing Htrp3S construct (Htrp3S: 0.12 ± 0.01; Htrp3AS: 0.17 ± 0.02). Therefore, to determine the true SOCE based on Ba2+ influx, we subtracted the basal Ba2+ leak from the total Ba2+ influx following store depletion. When the time course data are combined for the 36 Htrp3S coverslips and for the 32 Htrp3AS coverslips, we can see that the leak-subtracted Ba2+ influx (or SOCE) was greater in cells expressing the Htrp3S construct in comparison with cells expressing the Htrp3AS construct (Fig. 3C). To perform a statistical analysis of these two cell populations, we determined the SOCE for individual coverslips and averaged the values for each population. Based on this analysis, expression of Htrp3AS reduced SOCE by 32%, a difference which was statistically significant (Htrp3S: 0.56 ± 0.046; Htrp3AS: 0.38 ± 0.021, P < 0.0001; Fig. 3D). The reduction in SOCE by expression of Htrp3AS suggests that Htrp3 plays a functional role in mediating SOCE.

Suppression of SOCE in Htrp3AS cells transiently transfected with Htrp1 antisense construct. To assess the functional role of Htrp1 in SOCE in HEK-293 cells, we transiently transfected the Htrp1 antisense construct into the Htrp3AS stable cell line. We refer to these cells as Htrp3&1AS cells. For a control, we transiently transfected a short Htrp1 sense construct into the Htrp3S stable cell line. We refer to cells treated in this manner as Htrp3&1S cells. Forty-eight hours after transfection, both cell lines were monitored using fura 2 image analysis, and SOCE was estimated by addition of Ba2+ into Ca2+-free solution following thapsigargin treatment. We measured Ba2+ influx before and after Ca2+ store depletion (Fig. 4A) for both Htrp3&1S and Htrp3&1AS transfected cell lines and subtracted the basal leak of Ba2+ from the Ba2+ entry measured after store depletion to obtain a value for the SOCE. Whereas coexpression of antisense constructs for Htrp1 and Htrp3 had no measurable effect on the size of the internal Ca2+ store and its level of release (Fig. 4B), it did cause a dramatic decrease in SOCE (Fig. 4C). The magnitude of the effect of coexpression of Htrp1 and Htrp3 on SOCE was a 55% reduction in comparison with control cells (Fig. 4D). The data in Fig. 5 provides a direct comparison of the inhibition seen for expression of Htrp3AS alone and inhibition seen for coexpression of Htrp3&1AS constructs. The increase in inhibition (from 32% to 55%) obtained by transiently expressing Htrp1AS on top of the stable expression of Htrp3 is statistically significant (P < 0.05).


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Fig. 4.   Additional suppression of SOCE in Htrp3AS cells transiently transfected with an Htrp1 antisense construct. Htrp1 sense or Htrp1 antisense constructs were transiently transfected into Htrp3S or Htrp3AS cells (Htrp3&1S and Htrp3&1AS, respectively). Forty-eight hours after transfection, both cell lines were loaded with fura 2 and subjected to Ca2+ image study. Two millimolars Ba2+ was added to cells in a Ca2+-free solution both before and after thapsigargin treatment. Each Ca2+ trace shown represents response of a single coverslip with response averaged over ~800 cells. A: measurement of Ba2+ influx before and after Ca2+ store depletion with thapsigargin. B: comparison of thapsigargin-induced Ca2+ release in Htrp3&1S (fine line) and Htrp3&1AS (thick line) in the absence of Ca2+. Cells were perfused with Ca2+-free HHBSS and thapsigargin (1 µM) was added into chamber. C: comparison of SOCE in Htrp3&1S (fine line) and Htrp3&1AS (thick line) cell lines. After thapsigargin treatment, SOCE was determined by taking the difference between Ba2+ leak influx and Ba2+ influx following thapsigargin treatment. D: statistical analysis of SOCE in Htrp3&1S cells in comparison with Htrp3&1AS cells. Mean value of SOCE obtained for Htrp3&1S cell line (n = 10) was plotted as 100%. SOCE of Htrp3&1AS cell line (n = 9) was decreased by 55%. * Statistically significant difference from Htrp3&1S (P = 0.009).



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Fig. 5.   Statistical analysis of changes in SOCE between the four cell lines. Htrp3S cells or Htrp3AS cells were transiently transfected with either an Htrp1 sense construct (Htrp3&1S) or an Htrp1 antisense construct (Htrp3&1AS), respectively. Cell lines were loaded with fura 2 and SOCE was determined by subtracting Ba2+ leak flux from Ba2+ influx following store depletion with thapsigargin. Mean value of SOCE obtained in the Htrp3S cell line was plotted as 100%. In comparison, the SOCE of Htrp3AS cell line was decreased by 32%. In comparison with Htrp3&1S cells, the SOCE of Htrp3&1AS cells was reduced by 55%, which represented a statistically significant difference (P = 0.009). * Statistically significant difference from Htrp3S (P < 0.0001); ** statistically significant difference from Htrp3AS (P = 0.04).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using RT-PCR strategies, we tested for endogenous expression of trp homologues in fibroblasts from wild-type and src-/- mice and in HEK-293 cell lines. We found that all three cell lines express endogenous trp homologues although the levels of expression of individual trp homologues appear to differ from cell line to cell line. Three trp homologues (Mtrp2, Mtrp3, and Mtrp4) were detected in mouse fibroblasts and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) were detected in HEK-293 cells (Fig. 1). This is consistent with previous studies that mammalian trp transcripts are widely, but differently, expressed across tissues and cell lines (15, 30, 42, 53, 55). In addition, there can sometimes be differences in expression in the same cell type reported from different laboratories. These can sometimes be explained on the basis of differences in technical details between the way the PCR is run in the two laboratories. For example, a previous report of Htrp isoform expression in HEK-293 cells indicated that Htrp1 and Htrp6 were expressed at low levels in comparison with Htrp3 and Htrp4 (15), whereas our studies reported the strongest expression for Htrp1 and Htrp3, and although the expression for Htrp6 was lower than that of Htrp1 and Htrp3, it was roughly equal to that of Htrp4. One key difference in the way these experiments were run is that we used primers based on the human sequence of Htrp1 and Htrp6, whereas mouse primers were used in the other study (15). In our initial studies, we also used primers based on the mouse trp6 sequence and found a barely detectable band after 35 rounds of PCR (data not shown). When we changed to primers based on the human sequence, a very strong band was seen after 35 rounds of PCR (Fig. 1D). Thus the use of primers based on human sequence showed a more robust expression of the four Htrps in HEK-293 cells. The diversity of expression of multiple trp isoforms in mammals may provide a flexibility in the types of Ca2+ responses following stimulation of different cell types.

In HEK-293 cells in which the 323 bp Htrp3S construct was stably transfected, Northern blot analysis detected an ~4 kb Htrp3 mRNA transcript, whereas in HEK-293 cells stably transfected with a 323 bp Htrp3AS, this transcript was difficult to detect (Fig. 2), suggesting that endogenous expression of Htrp3 was dramatically suppressed. Furthermore, in HEK-293 cells in which the Htrp3S construct was expressed, SOCE was activated by thapsigargin via depletion of [Ca2+]i stores. However, in the Htrp3AS cells in which very little Htrp3 mRNA was expressed, the thapsigargin-induced SOCE was diminished significantly (Fig. 3D). One concern in performing stable transfections is that when comparing a limited number of control and experimental clones, one will obtain an artifactual result due to normal clonal variation of the measured parameter in the parental population (2a). We circumvented this problem by not selecting individual clones from the transfected population, but rather we combined all of the clones surviving the G418 selection. Our recent statistical analysis of the impact of clonal variation on this process (2a) indicates that if one averages the data over 200 clones from the transfected population, then one will have less than a 0.0001% chance of obtaining the 32% reduction in SOCE entry observed in our study, based purely on chance selection of clones which had lower SOCE before transfection. Thus the observed 32% reduction in SOCE in Htrp3AScells clearly indicates that in HEK-293 cells, endogenous Htrp3 is required for the full activity of SOCE after thapsigargin stimulation.

To investigate the involvement of Htrp1 in SOCE, we expressed the 369 bp Htrp1AS construct transiently on top of the Htrp3AS being stably expressed in HEK-293 cells. Transient expression of 369 bp Htrp1 sense construct in Htrp3S cells served as the control for these experiments. The transient expression of Htrp1AS served to increase the inhibition in Htrp3AS cells from 32% to 55%, an increase that was statistically significant (Fig. 5). Because the transient transfection would incorporate Htrp1AS into only about 60% of the Htrp3AS cells, and the measurements of Ba2+ entry were monitored over a field that would contain both transfected and untransfected cells, it is likely that the estimated combined contribution (55% of total) of Htrp1 and Htrp3 to SOCE entry is an underestimate. However, these results certainly support a role for these two trp homologues in mediating SOCE.

Functional analysis of mammalian trp homologues has received much attention recently; however, results obtained remain controversial. For almost all trp homologues identified, at least one study indicates that they function as SOCs, whereas there is also at least one study that argues that they function in a manner independent of store depletion. For example, although trp1 is store operated when expressed in mammalian cells (55, 57), it is constitutively active in the insect cell line, Sf9 cells (43). In HEK-293 cells in which cDNA of rabbit trp5 was stably transfected, Ca2+ influx was activated by Ca2+ store depletion by thapsigargin (38). In HEK-293 cells in which mouse trp5 was transiently transfected, Ca2+ influx following thapsigargin treatment was not enhanced in comparison with the control cells (33). Furthermore, when rat trp6 was transiently transfected into COS cells by electroporation, SOCE was activated following depletion of [Ca2+]i store by thapsigargin (28). However, when mouse trp6 was transiently expressed in COS-M6 cells by DEAE-dextran/chloroquine shock method, it did not significantly affect Ca2+ influx induced by thapsigargin, although Ca2+ entry stimulated by a G protein-coupled receptor was enhanced (6). In addition, when the cDNA coding for human trp6 was microinjected into the nuclei of CHO-K1 cells, it was observed that the channel was not selective for Ca2+ over Na+ or K+ (19). This current was directly activated by diacylglycerol (DAG) independently of protein kinase C activation or depletion of [Ca2+]i stores by perfusion with InsP3, suggesting DAG as an alternative regulator of opening trp channels. The importance of DAG was also emphasized in a recent study in Drosophila, where DAG metabolites, linoleic and linolenic acids, two members of the polyunsaturated fatty acids family, directly activated trp channels (9).

Similar confusion is observed when comparing the result of expression of trp3 in different cell lines by different methods. Expression of rat trp3 in COS cells (transiently by electroporation) revealed an enhance-ment of Ca2+ entry in response to the depletion of [Ca2+]i stores induced by thapsigargin (28). Likewise, expression of human trp3 in COS cells (transiently by DEAE-dextran/chloroquine shock) also led to a substantial increase in Ca2+ entry following stimulation of the muscarinic receptor with carbachol or when Ca2+ store was depleted with thapsigargin (55). Contrarily, expression of human trp3 cDNA insert in CHO cells (transiently by intranuclear microinjection) produced cation current that was constitutively active, with little selectivity for Ca2+ over Na+, and it was not enhanced in response to depletion of Ca2+ stores with thapsigargin or InsP3 (56). Furthermore, stable expression of human trp3 in HEK-293 cells (by Ca2+ phosphate/glycerol shock) gave rise to Ca2+ entry that was lower in response to thapsigargin than in response to carbachol, and addition of carbachol to thapsigargin-treated Htrp3 cells induced a further enhancement of Ca2+ influx (54). Finally, a recent study of HEK-293 cells stably expressing human trp3 verified Htrp3 as being store-operated channels (23).

How then does one reconcile all of the conflicting data on whether or not expression of trp homologues enhances SOCE in various cell types? More importantly for this paper, is there an explanation for why some of the data on Htrp3 expression support our results using the antisense knockout approach, whereas other data are in opposition to our conclusions? Perhaps the simplest explanation is that trp-encoded channels exist as tetrameters (5). Therefore, they have the potential to form either homotetramers or heterotetramers. In the COS cell line, exogenous Htrp3 might assemble with other trps to form a heterotetramer that could be activated by store depletion following thapsigargin. In the CHO cell line, however, exogenous Htrp3 might form a homotetramer, and its activation by store depletion might require another trp protein that is missing in CHO cells. Therefore, within the structural constraints of some cells, Htrp3 may not be able to form SOCs. In addition, the transfection method may have potential impact on the channel property. In comparison with antisense cDNA approach, the overexpression of full-length cDNA may provide misleading information if the trp is highly overexpressed, favoring formation of homotetrameric channels that may differ from the tetrameric nature of channels formed by endogenous, lower levels of Htrp3. For example, it has been shown that coexpression of Dtrp and Dtrpl in oocytes produces a current distinct from that resulting from expression of either trp or trpl alone (52). This may be the explanation for why the same trp (i.e., Htrp3) expressed in the same cell line (HEK-293 cell) can give rise to completely different results by the overexpression method (54) than is seen with the antisense cDNA approach we presented in this study. A further complication is that there can be a great deal of variation between populations of HEK-293 cells between different laboratories with endogenous levels of SOCE varying over a wide range. This could mean that levels of endogenous trp homologues could vary in HEK-293 cells in different laboratories.

Using "knockout" models raises a possibility that elimination of Htrp1 and Htrp3 may result in compensatory changes by other trp homologues or by an as yet unidentified SOCE component. Thus the level of reduction in SOCE observed in this study could turn out to be an underestimate, and not a true reflection of the extent of involvement of Htrp1 and Htrp3 in SOCE, due to reasons other than the fact that the Htrp1 transfection would not occur in every cell measured. This is a common problem which applies to all knockout models. However, regardless of the exact quantitation of the Htrp1 and Htrp3 contribution to SOCE, the present study provides strong support for the participation of Htrp1 and Htrp3 in SOCE.

In summary, depletion of internal Ca2+ stores has been causally linked to the activation of plasma membrane SOCs. The molecular basis and regulation of the underlying Ca2+ channel is still unknown, but it has been proposed to be a protein encoded by trp. The present study shows the identification of four human trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in HEK-293 cells, and the involvement of two of these homologues, Htrp 1 and Htrp3, in SOCE. It is our plan to investigate the tyrosine phosphorylation levels of these trp proteins to determine whether they are the downstream targets of tyrosine kinases in the regulation of SOCE.


    ACKNOWLEDGEMENTS

We thank Drs. Xian-Zhong Shawn Xu and Craig Montell, Johns Hopkins School of Medicine, for providing us with information on the unpublished sequence of Htrp4 for the design of our Htrp4 primers. We also thank Dr. Louis Philipson and his graduate student, Feng Qian, Medicine Department, University of Chicago, for providing mouse primers and useful advice for some of our preliminary studies.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-54500.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Dr. M. L. Villereal, Dept. of Neurobiology, Pharmacology and Physiology, Abb 532, Univ. of Chicago, 947 E. 58th St., Chicago, IL 60637 (E-mail: mitch{at}drugs.bsd.uchicago.edu).

Received 17 June 1999; accepted in final form 30 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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