Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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,
HCO3-free HBSS, and DMEM from Life
Technologies GIBCO BRL; and [
-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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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 [
-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,
HCO3-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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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