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J. Biol. Chem., Vol. 277, Issue 24, 21617-21623, June 14, 2002

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Comparison of Human TRPC3 Channels in Receptor-activated and Store-operated Modes

DIFFERENTIAL SENSITIVITY TO CHANNEL BLOCKERS SUGGESTS FUNDAMENTAL DIFFERENCES IN CHANNEL COMPOSITION^*

Mohamed Trebak, Gary St. J. Bird, Richard R. McKay, and James W. Putney Jr.

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, March 15, 2002, and in revised form, April 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd³⁺ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 µM Gd³⁺ or 30 µM 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 µM Gd³⁺ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (~60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 µM Gd³⁺ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd³⁺ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 µM. Finally, Gd³⁺ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd³⁺ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Calcium signaling plays a central role in regulating many physiological processes such as muscle contraction, cellular proliferation and differentiation, neurotransmitter secretion, and apoptosis. In a variety of nonexcitable cells, calcium signaling is initiated through cell membrane receptors coupled to phospholipase C, resulting in production of inositol 1,4,5-trisphosphate (IP₃)¹ (1). IP₃ releases intracellular Ca²⁺ from the endoplasmic reticulum. This release of intracellular Ca²⁺ triggers an influx of Ca²⁺ across the plasma membrane, a process known as capacitative calcium entry (CCE) or store-operated calcium entry (2-5). The mechanism underlying the activation of plasma membrane calcium-permeable channels is not fully understood, but the initiating signal is the depletion of endoplasmic reticulum calcium content. Currents mediated by store-operated channels have been measured in various cell types (6). The best defined store-operated current to date is the Ca²⁺ release-activated Ca²⁺ current (I_crac), found predominantly in hematopoietic cells (6, 7). I_crac measured in mast cells, Jurkat cells, and rat basophilic leukemia cells is both highly selective for Ca²⁺ and completely blocked by low concentrations of lanthanides (1 µM Gd³⁺). However, in many other cell types, store-operated currents with moderate divalent ion selectivity have been recorded (8, 9), as well as nonselective cation currents (10, 11).

Although CCE has been widely studied in many different cell types, the molecular identity of store-operated channels (SOCs) and the signal by which store emptying activates those channels remain uncertain. Two major hypotheses about the mechanism of CCE have been proposed. The first proposes the release of a putative calcium influx factor from the endoplasmic reticulum (12, 13). The second, the "conformational coupling" model, involves direct interaction of IP₃ receptors in the endoplasmic reticulum with SOCs in the plasma membrane (3, 14). Mammalian homologues of the Drosophila transient receptor potential (TRP) channel are candidates for store-operated calcium channels. Among the canonical TRP subfamily (designated TRPC1 through TRPC7), human TRPC3 (hTRPC3), first cloned by Zhu et al. (15), has been shown in many heterologous expression systems, including HEK293, to behave as a receptor-activated channel with constitutive activity that cannot be further increased by store depletion (15-18). Furthermore, Hofmann et al. (16) showed that TRPC3 and its structural relative TRPC6 can be activated by diacylglycerol, providing a possible mechanism of activation of these channels by phospholipase C-linked receptors, independently of IP₃ and store depletion. On the other hand, despite the demonstrated absence of regulation of hTRPC3 by store emptying in these expression systems, a substantial body of evidence suggests that hTRPC3 activation involves interaction with IP₃ receptors (IP₃Rs) or ryanodine receptors consistent with the conformational coupling model (19-21).

This laboratory recently demonstrated that hTRPC3 is regulated by store depletion when transiently expressed in DT40 chicken B-lymphocytes (22). A substantial increase in Ba²⁺ entry was observed in hTRPC3-DT40 cells after store depletion by thapsigargin. Moreover, the store depletion activity of hTRPC3 expressed in DT40 cells partly depended on the presence of IP₃Rs. Therefore, we proposed hTRPC3 as a candidate for store-operated, Ba²⁺-permeable channels, perhaps similar to those observed in nonhematopoietic cells.

The differences between hTRPC3 channels generated in HEK293 cells versus those generated in DT40 cells are intriguing. Thus, we have undertaken a comparison of the sensitivity of hTRPC3 channels expressed in either HEK293 cells or DT40 cells, as well as their endogenous CCE channels, to the actions of two well characterized inhibitors of CCE. Gd³⁺, at least when employed in the low micromolar range, is a relatively specific blocker of store-operated channels (23, 24) and is presumed to block through direct binding to sites in the channel pore. 2-Aminoethoxydiphenyl borane (2APB) appears to block store-operated channels through a direct action (25-29) but also appears capable of inhibiting conformationally coupled TRPC3 in HEK293 cells by virtue of an action on intracellular IP₃ receptors (17). Our findings show that receptor activation of hTRPC3-mediated cation entry in HEK293 cells is partially inhibited by 2APB, and the store-operated entry in DT40 cells is slightly reduced. This is consistent with the suspected involvement of IP₃ receptors in these two cell types. A surprising finding was that, in contrast to HEK293 cells, hTRPC3-mediated store-operated cation entry in DT40 cells is completely blocked by low concentrations of Gd³⁺. This finding reveals a fundamental difference in the molecular structure of hTRPC3 channels in these two cell lines, and this difference seems likely to be related to the two distinct modes of activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Thapsigargin and methacholine were purchased from Calbiochem. 2APB was synthesized as described previously (30).

Cell Culture and Transfection-- HEK293 cells were obtained from ATCC and were transfected, using Superfect reagent (Qiagen) according to the vendor's instructions, with pcDNA3 vector containing the green fluorescent protein (GFP) coding sequence added in frame to the C terminus of hTRPC3 (18). Cells stably expressing hTRPC3-GFP fusion protein were selected first by antibiotic resistance and second by GFP fluorescence by flow cytometry. Cells were grown under selection with G418 at 37 °C in Dulbecco's modified eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine in a humidified 95% air, 5% CO₂ incubator. The GFP fusion of hTRPC3, stably transfected into HEK293 cells, recapitulates the behavior of the wild-type channel in every aspect known for this channel: formation of a Ba²⁺- and Ca²⁺-permeable channel, constitutive activity, activation by agonist, activation by diacylglycerol, lack of activation by thapsigargin, sensitivity to 2APB, and insensitivity to low concentrations of Gd³⁺ (results of this study and data not shown (for diacylglycerol), compared with previously published findings from this (18) and other (15, 19) laboratories (17, 18)). Cell-attached patch clamp experiments² with the hTRPC3-GFP transfected HEK293 cells revealed the presence of single channels with similar conductance and kinetic behavior (appearance of subconductance states) and similar increase in open probability in response to carbachol as reported for wild-type hTRPC3 (19).

The immortalized avian B lymphocyte cell line, DT40 (Institute of Physical and Chemical Research (RIKEN) cell bank no. RCB1464) was kindly provided by Dr. Tomohiro Kurosaki (Kansai Medical University, Kansai, Japan). DT40 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% chicken serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol at 40 °C. Approximately 5 × 10⁶ DT40 cells were harvested from flasks containing about 5-8 × 10⁴ cells/ml and then transiently transfected by electroporation using a Gene Pulser apparatus (Bio-Rad) with 10 µg of the human isoform of TRPC3 (hTRPC3 in pcDNA3 vector, provided by Dr. Lutz Birnbaumer, NIEHS, National Institutes of Health), along with 5 µg of peYFP-C1 vector (CLONTECH) as a marker for transfection. Cells were assayed 18-30 h post-transfection.

Measurement of Intracellular Calcium-- For wild-type and hTRPC3-expressing HEK293 cells, calcium measurements were performed on attached populations of cells in polylysine-coated 96-well plates with a fluorometric imaging plate reader (FLIPR³⁸⁴; Molecular Devices, Inc., Sunnyvale, CA), except when stated otherwise. Cells were cultured to about 70% confluence, and 60,000 cells/well were plated and used 24 h after plating. Cells were loaded with either of two single visible wavelength indicators: for Ca²⁺, Fluo-4 (4 µM Fluo-4/AM for 45 min at 37 °C in complete Dulbecco's modified Eagle's medium), excited at 488 nm, and emission-selected by 510-570-nm bandpass filter; for Ba²⁺, Calcium Green-1 (4 µM Calcium Green-1/AM for 120 min at room temperature in a Hepes-buffered physiological saline solution (HBSS; 140 mM NaCl, 4.7 mM KCl, 1 mM MgCl₂, 1.0 mM CaCl₂, 10 mM glucose, 10 mM Hepes, pH 7.4) with 250 µM probenecid and 0.2% pluronic acid). In both cases, cells were washed twice in nominally Ca²⁺-free (i.e. no added Ca²⁺) HBSS. Detection was performed with a cooled charge-coupled device camera. Experiments were carried out at room temperature.

For calcium measurement experiments with DT40 cells, the low transfection efficiency makes it impractical to use the FLIPR³⁸⁴-based Ca²⁺ assay. DT40 cells were allowed to attach to glass coverslips for 15 min at 40 °C and loading was performed with 2 µM Fura-2/AM for 30 min at room temperature as described earlier (22). The cells were then washed three times and bathed at room temperature for at least 15 min before Ca²⁺ measurements. Experiments were initiated in a nominally Ca²⁺-free medium, which was identical in composition except for the omission of CaCl₂. Fluorescence measurements were performed under the conditions indicated with single enhanced yellow fluorescent protein-positive cells, which were selected for their fluorescence when excited at 488 nm, and emission wavelength was observed at 520 nm. Previous work has indicated that the efficiency of co-transfection of DT40 cells with two constructs with the methods used in this study is close to 100% (22). The fluorescence of Fura-2-loaded DT40 cells was monitored with a photomultiplier-based system, mounted on a Nikon Diaphot microscope (40× Neofluor objective). The cells were excited alternatively by 340- and 380-nm wavelength light from a filter-based illumination system (Photon Technology International, Princeton, NJ). Fluorescence emission at 510 nm was recorded by a photomultiplier tube (Omega Optical). All experiments were conducted at room temperature. The data are expressed as the ratio of Fura-2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of Gd³⁺ and 2APB on Ca²⁺ Entry in Wild-type and hTRPC3-transfected HEK293 Cells-- The generation of a stable population of hTRPC3-transfected HEK293 cells makes it practical to investigate Ca²⁺ signaling mechanisms utilizing the real time fluorescence-imaging plate reader system, FLIPR. In these experiments, background controls (i.e. Ca²⁺ additions in the absence of agonist or thapsigargin) were carried out in parallel for both wild-type and transfected cells. The background data are summarized in Fig. 1 and indicate that hTRPC3 produces a constitutive Ca²⁺ entry, consistent with previous findings (15, 18). Interestingly, this entry was partly blocked by Gd³⁺ and blocked to a greater extent by 2APB (see also Ref. 27). Fig. 2 summarizes the results of experiments examining the sensitivity to Gd³⁺ and 2APB of Ca²⁺ entry in response to either agonist (300 µM methacholine) or thapsigargin (2 µM) activation in wild-type and hTRPC3-transfected cells. The background data from experiments of Fig. 1 have been subtracted. The results in the wild-type cells are similar to those reported previously for this cell line (31); both Gd³⁺ and 2APB caused essentially complete block of both agonist- and thapsigargin-induced Ca²⁺ entry. For the case of the hTRPC3 cells, somewhat unexpected results were obtained. With agonist activation, there was no significant effect of Gd³⁺ on Ca²⁺ entry, and 2APB reduced only the peak of the Ca²⁺ entry response. Additionally, the hTRPC3 cells exhibited a thapsigargin-activated Ca²⁺ entry that was partially resistant to inhibition by Gd³⁺ or 2APB.³

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of Gd3+ (3 µM) and 2APB (30 µM) on basal influx of Ca2+ in wild-type (top) and hTRPC3-transfected (bottom) HEK293 cells. Cells were incubated in the absence of added Ca2+, and 1.8 mM Ca2+ was restored where indicated. At the first arrow, additions were as follows: vehicle (control, no inhibitor) (solid line), 3 µM Gd3+ (dotted line), and 30 µM 2APB (dashed line). Curves are averages of three independent experiments, each involving four wells per condition, carried out utilizing the FLIPR384 real time plate scanner.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effect of Gd3+ (3 µM) and 2APB (30 µM) on methacholine-activated (top two panels) and thapsigargin-activated (bottom two panels) influx of Ca2+ in wild-type (left two panels) and hTRPC3-transfected (right two panels) HEK293 cells. Cells were incubated in the absence of added Ca2+, and 300 µM methacholine (MeCh) or 2 µM thapsigargin, followed by 1.8 mM Ca2+ was added where indicated. At the first arrow, additions were as follows: vehicle (control, no inhibitor) (solid line), 3 µM Gd3+ (dotted line), and 30 µM 2APB (dashed line). Curves are averages of three independent experiments, each involving four wells per condition, carried out utilizing the FLIPR384 real time plate scanner.

The results with agonist activation present something of a paradox; methacholine induces complete depletion of thapsigargin-sensitive Ca²⁺ stores,⁴ yet it does not appear to activate any Gd³⁺-sensitive store-operated entry. Also, previous studies have indicated that the agonist activation of hTRPC3 is blocked by 2APB, presumably reflecting a role of IP₃ receptors (17). Finally, the results with thapsigargin could be interpreted to indicate that hTRPC3 forms Ca²⁺-permeable, store-operated channels, despite previous conclusions that only noncapacitative channels are formed in HEK293 cells (15, 17, 18).

One possible explanation for these unexpected findings is that they arise from complex mechanisms of Ca²⁺ regulation known to occur in most cell types. In the case of responses to agonist, it is possible that activation of plasma membrane pumps by Ca²⁺ and feedback inhibition of the store-operated channels by Ca²⁺ limit the extent of the [Ca²⁺]_i rise when both capacitative and noncapacitative mechanisms operate simultaneously. The transient rise in [Ca²⁺]_i in thapsigargin-treated hTRPC3 cells could reflect store-operated channels; on the other hand, this could as easily reflect the constitutive activity of hTRPC3 channels, exaggerated by the inability (due to thapsigargin) of intracellular endoplasmic reticulum to efficiently buffer entry through these channels.

To avoid complications of alterations in Ca²⁺ transport and Ca²⁺ regulation of channels, we next carried out experiments utilizing Ba²⁺ as a surrogate for Ca²⁺. The use of Ba²⁺ avoids many of these problems, since it generally does not activate Ca²⁺-sensitive regulatory sites and is a poor substrate for Ca²⁺ transport mechanisms (32-35). Fig. 3 illustrates Ba²⁺ (1 mM) entry data obtained with FLIPR analysis of both wild-type and hTRPC3 HEK293 cells. As Ba²⁺ accumulates in the cell, complex and unknown mechanisms may limit its maximum level of accumulation. Thus, rather than utilizing the maximum value for Ba²⁺ entry, we calculated the initial rates of Ba²⁺ entry with the different modes of activation and in the two cell types (summarized in Fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of methacholine (MeCh) on influx of Ba2+ in wild-type (left) and hTRPC3-transfected (right) HEK293 cells. Cells were incubated in the absence of added Ca2+, and 300 µM methacholine (MeCh) and 1 mM Ba2+ were added where indicated. Dotted line, control (no methacholine); solid line, 300 µM methacholine. Curves are averages of seven (wild type) and eight (hTRPC3-transfected) independent experiments, each involving four wells per condition, carried out utilizing the FLIPR384 real time plate scanner.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of Gd3+ (3 µM) and 2APB (30 µM) and both inhibitors in combination on methacholine- and thapsigargin-activated influx of Ba2+ in wild-type (top) and hTRPC3-transfected (bottom) HEK293 cells. Cells were incubated in the absence of added Ca2+, and 300 µM methacholine (MeCh, black bars) or 2 µM thapsigargin (TG, hatched bars) followed by 1 mM Ba2+ was added following the protocol shown in Fig. 3. Initial rates of Ba2+ entry (fluorescence increase upon the addition of Ba2+) were calculated. Presented are means ± S.E. from seven (wild-type) and eight (hTRPC3-transfected) independent experiments, each involving four wells per condition.

As seen in the Ca²⁺ experiments, the basal rate of Ba²⁺ entry was significantly greater in hTRPC3 cells than in wild-type cells (Fig. 4). This statistically significant difference was seen also in the presence of Gd³⁺, in the presence of 2APB, or both (p < 0.05). When the Gd³⁺-insensitive Ba²⁺ entry, which should contain the contribution of hTRPC3, was subtracted from the basal rates, the remaining Gd³⁺-sensitive component was not significantly different in the two cell lines (rates: control, 5.9 ± 0.4 cps²; TRPC3, 5.7 ± 0.5 cps²). Thus, the increase in basal entry appears to result from constitutive activity of hTRPC3 rather than from a small activation of a store-operated pathway.

In wild-type HEK293 cells, both methacholine and thapsigargin activated Ba²⁺ entry and to approximately the same extent, as expected if both agents are acting through store depletion. This entry was essentially completely blocked by either Gd³⁺ or 2APB, as expected from previous findings (31) and from the results in Fig. 2. In hTRPC3 cells, Ba²⁺ entry in response to methacholine was clearly greater than in the wild-type cells, but the response to thapsigargin was not significantly different. As in the wild-type cells, the response to thapsigargin was completely blocked by either Gd³⁺ or 2APB. The response to methacholine in hTRPC3 cells was partially blocked by Gd³⁺, consistent with a co-activation of both capacitative and noncapacitative (hTRPC3-mediated) pathways. While 2APB did not produce a complete block of the agonist response, it produced a statistically greater effect than did Gd³⁺ and was able to further reduce entry in cells blocked by Gd³⁺. This is consistent with the view that Gd³⁺ blocks the capacitative component of the entry, whereas 2APB blocks the capacitative component, and at least partially inhibits activation of hTRPC3. Interestingly, the failure of 2APB to completely block the hTRPC3 response does not appear to result from an inadequate concentration (30 µM in this experiment), since concentration-effect studies revealed no additional inhibitory effect with concentrations as high as 100 µM (data not shown).

Thus, the data with Ca²⁺ involve a number of potential artifacts; the Ba²⁺ data indicate that the apparent effect of thapsigargin in activating entry of Ca²⁺ through hTRPC3 channels results from thapsigargin-induced reduction of intracellular Ca²⁺ buffering by endoplasmic reticulum rather than an actual store-dependent regulation of hTRPC3. It is likely that a similar explanation underlies findings of altered basal Ca²⁺ kinetics in hTRPC3 cells in a previous study (15). Likewise, the failure of Gd³⁺ to inhibit the Ca²⁺ entry in hTRPC3 cells is not borne out by the Ba²⁺ data and thus probably results from nonlinear buffering of Ca²⁺ or perhaps Ca²⁺-mediated cross-talk between SOCs and hTRPC3 channels.

Gd³⁺ and 2APB Inhibit Capacitative Calcium Entry in DT40 Cells-- In Fig. 5, typical Fura-2 single cell experiments with wild-type DT40 cells are shown. Thapsigargin was added to the cells initially in a nominally Ca²⁺-free solution. After the Ca²⁺ release phase, Gd³⁺ or 2APB was added a few minutes before restoring Ca²⁺ to a concentration of 1.5 mM in the external medium. Similar to findings in HEK293 cells, the endogenous CCE in DT40 cells was essentially completely blocked by either Gd³⁺ or 2APB (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of Gd3+ and 2APB on capacitative calcium entry in DT40 cells. The cells were loaded with Fura-2, and single-cell calcium measurements were performed as described under "Materials and Methods." Experiments were initiated in Ca2+-free medium, and 2 µM thapsigargin and 1.8 mM Ca2+ were added where indicated. Ca2+ entry induced by 2 µM thapsigargin in wild-type DT40 cells was essentially blocked when 1 µM Gd3+ or 30 µM 2APB were added. Data are representative of three independent experiments.

Store-operated hTRPC3-mediated Cation Entry in DT40 Cells Is Inhibited by Gd³⁺ and Partially Inhibited by 2APB-- We reported previously that hTRPC3 behaves as a store-operated Ba²⁺-permeable channel when transiently expressed in the avian B cell line DT40 (22). Unlike the HEK293 expression system, wild-type DT40 cells do not exhibit thapsigargin-activated Ba²⁺ entry (22). Also, when hTRPC3 is expressed in DT40 cells, no constitutive permeability is detected with either Ca²⁺ or Ba²⁺ (22). However, store depletion with thapsigargin induces a significant Ba²⁺ entry that is observed in neither wild type nor mock-transfected cells. We have not as yet succeeded in producing stable, hTRPC3-expressing DT40 cells, and thus we have utilized transient transfection as described in our earlier report (22). Ba²⁺ entry in response to thapsigargin was assessed in single enhanced yellow fluorescent protein-positive cells using Fura-2 imaging as indicated under "Materials and Methods." Surprisingly, and in contrast to findings in HEK293 cells, the addition of 1 µM Gd³⁺ after the calcium release phase completely inhibited hTRPC3-mediated barium entry in response to thapsigargin in DT40 cells, whereas 30 µM 2APB had minimal effect (Fig. 6, but see Fig. 8 and discussion below).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of Gd3+ and 2APB on Ba2+ entry in hTRPC3-transfected DT40 cells. The protocol was identical to Fig. 5, except that 10 mM Ba2+ was added where indicated to cells previously transfected with hTRPC3, as described under "Materials and Methods."

We then investigated the concentration dependence of the inhibitory actions of Gd³⁺ on store-operated hTRPC3-mediated barium entry and on endogenous capacitative calcium entry. Fig. 7 shows that these two store-operated cation entries show similar sensitivity to Gd³⁺ inhibition, with a nearly complete block (90% or greater) at a concentration of 0.1 µM.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Capacitative cation entry in wild-type (filled symbols) and in hTRPC3-transfected DT40 cells (open symbols). Initial rates of either Ca2+ (for the wild-type cells) or Ba2+ (for the hTRPC3 cells) were measured after cells were loaded with Fura-2 and exposed to thapsigargin to deplete the stores in a similar protocol to previous figures. For endogenous CCE, 1.5 mM Ca2+ was restored to the external medium after the calcium release phase, whereas 10 mM Ba2+ was used in the case of hTRPC3-transfected cells to assess cation entry. Experiments were carried out in the absence or in the presence of the indicated concentrations of Gd3+. Data are means ± S.D. from 2-8 experiments.

Statistical analysis of a number of experiments with 2APB revealed that this reagent caused a small but statistically significant inhibition (~25%) of store-operated Ba²⁺ entry (Fig. 8). A similar degree of inhibition was observed with 75 and 100 µM 2APB, indicating that this is the maximal degree of inhibition obtainable with 2APB.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of 2APB on store-operated hTRPC3 activity in DT40 cells. The protocol was identical to that for Figs. 6 and 7 with 10 mM Ba2+ added to the external medium after depletion of the store by thapsigargin. 2APB was added at different concentrations, and the slope of the barium entry trace was determined. Data represent means ± S.E. from 3-9 different experiments.

Although store depletion clearly activates a Ba²⁺-permeable channel in hTRPC3-DT40 cells, it is not clear how significant such a channel might be as a mechanism for Ca²⁺ signaling. The ability of 2APB to block endogenous CCE completely with only a partial effect on hTRPC3 store-dependent activity in DT40 provides an opportunity to observe hTRPC3-dependent Ca²⁺ entry in isolation from the endogenous pathway. We used a protocol similar to the one in Fig. 5A except that 30 µM 2APB was included after the calcium release phase. When store depletion was induced by thapsigargin, a robust Ca²⁺ entry ensued, presumably mediated exclusively by hTRPC3 channels. The addition of 1 µM Gd³⁺ rapidly reversed this entry (Fig. 9).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Effect of Gd3+ on store-operated calcium entry mediated by hTRPC3 in DT40 cells. The protocol was similar to that for Fig. 5 except that 30 µM 2APB was added after the calcium release phase and kept throughout the experiment to block endogenous CCE. When store depletion is induced by thapsigargin, a robust calcium entry follows, which is presumably mediated exclusively by hTRPC3 channels. The addition of 1 µM Gd3+ rapidly reverses this calcium entry. Data are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transient expression of hTRPC3 in DT40 B lymphocytes generates a divalent cation-permeable channel that is activated by store depletion (22). Data from a number of laboratories including our own have shown that hTRPC3 transiently or stably expressed in HEK293 cells behaves as a receptor-operated cation channel that is not activated by store depletion (15, 17, 18). We thus investigated the actions of previously characterized inhibitors of store-operated channels, comparing the behavior of hTRPC3 expressed in DT40 cells with hTRPC3 expressed in HEK293 cells.

In investigations of hTRPC3 regulation in HEK293 cells, we discovered potential ambiguities and artifacts arising from measurements of Ca²⁺ entry. These are probably attributable to complex and nonlinear actions of Ca²⁺ transport, buffering, and negative feedback mechanisms. These problems were revealed and also alleviated by use of Ba²⁺, a surrogate for Ca²⁺, which passes through many types of Ca²⁺ channels but which is a poor substrate for Ca²⁺ pumps and a poor activator of Ca²⁺ feedback processes (32). Thus, expression of hTRPC3 results in an apparent entry of Ca²⁺ following depletion of stores with thapsigargin. However, despite the fact that TRPC3 channels are readily Ba²⁺-permeable, no such entry of Ba²⁺ was observed. The simplest explanation of this discrepancy is that the apparent entry of Ca²⁺ was a manifestation of constitutive activity of hTRPC3, amplified by thapsigargin's abrogation of intracellular buffering by the endoplasmic reticulum. Thus, caution should be used in studies involving expression of Ca²⁺ channels in which some channels become constitutively active. A similar problem occurred in studies of the action of inhibitors. Thus, Gd³⁺ appeared incapable of affecting agonist-activated Ca²⁺ entry in HEK293 cells, yet when Ba²⁺ entry was examined, about half of the activated entry was blocked. The latter result is consistent with the expectation that the agonist, by virtue of its ability to deplete endoplasmic reticulum Ca²⁺ stores, should activate a Gd³⁺-sensitive store-operated entry. The apparent failure of Gd³⁺ to inhibit Ca²⁺ entry under the same circumstances may result from a nonlinear activation of plasma membrane Ca²⁺ extrusion such that a limiting level of [Ca²⁺]_i is set. It is also possible that Ca²⁺ entering through hTRPC3 channels specifically suppresses the endogenous store-operated channels, which are known to be highly sensitive to inhibition by Ca²⁺. Regardless of the explanation, the use of Ba²⁺ as a surrogate for Ca²⁺ in HEK293 allows detection of both modes of entry.

In both cell types, transfection with hTRPC3 resulted in the expression of channels with properties clearly distinct from the endogenous channels. Thus, it is likely that hTRPC3 is at least a constituent of the newly formed channels in both instances. However, a significant finding in this study is that hTRPC3-dependent store operated cation entry in DT40 cells is blocked by Gd³⁺, whereas in HEK293 cells it is completely insensitive to this lanthanide. Lanthanides are generally believed to block channels by binding to sites in the channel pore (36, 37). Therefore, the molecular composition of the hTRPC3 channels in DT40 cells is probably different from hTRPC3 channels previously generated in other expression systems, including HEK293 cells. When hTRPC3 was overexpressed in different mammalian cell lines (15, 16, 38), it behaved as a receptor-operated nonselective cation channel that was not activated by store depletion. Specifically, hTRPC3 expressed in HEK293 cells was insensitive to inhibition by 10 µM Gd³⁺ (15), and Ohki et al. (39) succeeded in inhibiting TRPC3 expressed in Xenopus oocytes only when very high concentrations of Gd³⁺ (2 mM) were used. These data are consistent with our findings on hTRPC3 expressed in HEK293 cells.

Store-operated hTRPC3-mediated Ba²⁺ entry in DT40 cells shows a sensitivity to Gd³⁺ that is similar to the endogenous store-operated Ca²⁺ entry, with nearly complete block at concentrations as low as 0.1 µM (Fig. 7). In addition, Gd³⁺ inhibits fully established hTRPC3 activity (Fig. 9), suggesting that as for SOCs, Gd³⁺ acts directly on the hTRPC3 channels themselves. We previously speculated that a low expression level of hTRPC3 in DT40 cells is related to its regulation by store depletion (22). It is thus possible that the association of hTRPC3 with a limited quantity of endogenous components of CCE in the DT40 expression system determines the mode of coupling of hTRPC3 channels as well as the sensitivity to low concentrations of Gd³⁺. On the other hand, a high hTRPC3 expression level in HEK293 cells may result in homotetrameric hTRPC3 channels that would be insensitive to activation by store depletion and also insensitive to inhibition by low concentrations of lanthanides.

Some recent studies have provided evidence for the conformational coupling model for SOC activation (19, 20). According to this model, IP₃ receptors in the endoplasmic reticulum can sense Ca²⁺ depletion from the stores and convey a signal to SOCs in the plasma membrane via protein-protein interactions (3, 14). More recently, Ma et al. (17) provided data that were interpreted as supporting this conformational coupling model by use of 2APB, believed to act as a membrane-permeant IP₃ receptor antagonist. These authors showed that 2APB prevented both store-induced SOC activation and receptor-coupled hTRPC3 activation when hTRPC3 was expressed in HEK293 cells and concluded that IP₃Rs were essential for both SOC and hTRPC3 channel activation. However, in rat basophilic leukemia cells, 2APB blocked I_crac current completely when applied from the outside of the cells but inhibited only partially when applied from the cytoplasm, suggesting that 2APB may be acting as a direct blocker of SOC rather than as an IP₃ receptor antagonist (40). Kukkonen et al. (41) confirmed these findings with whole-cell patch clamp experiments in the same cells and reported that I_crac activity was rapidly blocked by extracellular 2APB, whereas intracellular 2APB was less effective. Furthermore, Broad et al. (25) showed that 2APB abolishes CCE induced by thapsigargin even in DT40 cells deficient for all isoforms of IP₃ receptor, consistent with a direct action of 2APB on the SOC channels themselves.

On the other hand, Ma et al. (17) showed that 2APB blocks TRPC3 channels when activated by a phospholipase C-linked agonist but not when activated with diacylglycerol. Thus, it is likely that the inhibitory effect of 2APB on hTRPC3 channels is not a direct effect but rather results from an action on IP₃Rs. We found that in our stable hTRPC3-expressing HEK293 cells, 2APB partially blocks receptor-induced hTRPC3 activity (~60% inhibition). We cannot definitively explain the quantitative difference between our results and those of Ma et al. (17). However, we note that our data are statistical averages obtained using cell populations of our own independently generated TRPC3-expressing cell line in a high throughput fluorescence system whereby hTRPC3 activity was assessed after stimulation with a maximal concentration of a cholinergic agonist. We also showed that 2APB even at high concentrations has a slight effect on store-operated hTRPC3 in DT40 cells (on average ~25% inhibition) that was statistically significant. In a previous study, we reported that store-dependent activation of TRPC3 channels in DT40 cells is about 50% diminished in a cell line lacking IP₃Rs, indicating that about half of the expressed channels depend on IP₃Rs for activation (22). Hence, the observed degree of inhibition is about what one would expect given that only half of the channels interact with IP₃ receptors and that, based on the results in HEK293 cells, we expect those channels to be only partially inhibited.

In conclusion, store-operated hTRPC3 channels in DT40 cells show similar sensitivity to low concentrations of Gd³⁺ as the endogenous SOCs. The correlation between store-dependent regulation of hTRPC3 and their inhibition by low concentrations of lanthanides is consistent with our supposition that hTRPC3 forms a component of SOCs by combining with unknown components in DT40 that also convey Gd³⁺ sensitivity. We also suggest that TRPC3 may be a constituent of a store-operated channel in nonhematopoietic cells in which store-operated channels are less Ca²⁺-selective. Clearly, further studies are needed to elucidate the contribution of hTRPC3 in this important pathway known as capacitative calcium entry.

	ACKNOWLEDGEMENTS

We are grateful for helpful comments by Dr. Elizabeth Murphy and Dr. Jerel Yakel. We thank Franz-Josef Braun for sharing unpublished findings on single channel properties of hTRPC3-GFP. We thank Dr. Lutz Birnbaumer (NIEHS, National Institutes of Health) for providing the cDNA for human TRPC3 and Dr. Tomohiro Kurosaki (Kansai Medical University, Kansai, Japan) for providing the DT40 cells.

	FOOTNOTES

^* 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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, P.O. Box 12233, MD:F2-02, Research Triangle Park, NC 27709. Tel.: 919-541-1129; Fax: 919-541-1898; E-mail: trebak@niehs.nih.gov.

Published, JBC Papers in Press, April 9, 2002, DOI 10.1074/jbc.M202549200

² F.-J. Braun, unpublished data.

³ The size of the Ca²⁺ release transient appears smaller in the hTRPC3 cells than in wild type, causing us to consider the possibility that hTRPC3 causes some depletion of Ca²⁺ pools. However, further investigation leads us to conclude that the difference in the magnitude of the apparent [Ca²⁺]_i signals in wild-type and TRPC3-transfected cells results from different loading or quantitative behavior of Fluo-4 in the two cell lines. For example, attempts to measure F_max by use of high concentrations of an ionophore gave a smaller maximum signal in the TRPC3 cells than in the wild-type cells. Additionally, the thapsigargin-induced Ca²⁺ entry appears less in the TRPC3 cells, but when Ba²⁺ is used (see Fig. 4), this difference is not seen (control rate, 11.5 ± 0.6; TRPC3-transfected rate, 13.3 ± 2.7). To resolve this issue more quantitatively, we carried out some additional experiments using an imaging system with which we could use a ratioing dye (Fura-2) that could be calibrated. With this technique, the peak [Ca²⁺]_i values following thapsigargin addition to the different cell lines were not statistically different: wild-type, 258 ± 32 nM; TRPC3, 272 ± 33 nM (n = 3). Thus, there does not appear to be any significant pool depletion associated with expression of TRPC3.

⁴ M. Trebak, G. St. J. Bird, R. R. McKay, and J. W. Putney, Jr., unpublished observation.

	ABBREVIATIONS

The abbreviations used are: IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; CCE, capacitative calcium entry; TRP, transient receptor potential; hTRPC3, human transient receptor potential gene product; SOC, store-operated channel; I_crac, calcium release-activated calcium current; GFP, green fluorescent protein; 2APB, 2-aminoethoxydiphenyl borane; FLIPR, fluorometric imaging plate reader; HBSS, Hepes-buffered physiological saline solution.

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