From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709
Received for publication, March 3, 2003
, and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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We recently demonstrated that TRPC3 is regulated by store depletion when transiently expressed in DT40 chicken B-lymphocytes (14), and we proposed TRPC3 as a candidate for store-operated, non-selective cation channels. However, Venkatachalam et al. (15) reported that, in this same cell line, TRPC3 behaves as a receptor-activated channel with no dependence on the depletion of Ca2+ stores. In addition, the store-operated behavior of TRPC3, which we have described previously (14), conflicts with the non-store-operated, receptor-regulated behavior reported by us for HEK293 cells (12). We thus suggested that the mode of activation of TRPC3 channels may depend on the level of expression (14). In the present work, we addressed directly the impact of channel expression levels on the mechanism of regulation of TRPC3 in the avian B cell line, DT40.
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MATERIALS AND METHODS |
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DT40 cells were transiently transfected by electroporation (14) with the indicated amounts of the human isoform of TRPC3 (TRPC3 into pcDNA3 vector, provided by Lutz Birnbaumer, NIEHS) or its vector (pcDNA3) along with the EYFP-C1 vector (Clontech) as a marker for transfection. When indicated, cells were co-transfected with the human M5 muscarinic receptor (50 µg/ml in pcDNA3) and/or rat IP3R-3 in pCB6+ (10 µg/ml; provided by Dr Graeme Bell, University of Chicago, Chicago, IL). For some experiments, TRPC3 was subcloned into the lcf201 expression vector (provided by Drs Jean-Marie Buerstedde and Hiroshi Arakawa, Heinrich-Pette-Institute, University of Hamburg, Germany) under the control of the chicken -actin promoter for expression in DT40 cells. Cells were assayed 1725 h post-transfection. Fluorescence measurements were performed under the conditions indicated with single enhanced yellow fluorescent protein (EYFP) positive cells, selected by their yellow/green fluorescence (excitation, 485 nm; emission, 520 nm). The fluorescence intensity of multiple Fura-2-loaded DT40 cells was monitored with a CCD camera-based imaging system (17). Basal Ca2+ levels in both wild-type and IP3 receptor knock-out (IP3R-KO) (16) DT40 cells were similar (around 110135 nM). Under the conditions of measurement, EYFP expression did not contribute significant fluorescence. In Figs. 2, 3, 4, 5, average traces from 814 EYFP-positive cells are shown for a single experiment that is representative of at least three independent experiments. The total number of cells responding for all experiments is mentioned in the text.
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To generate the TRPC3-Topaz fusion construct (T3T), TRPC3 was fused to the brighter EYFP version Topaz (18) at the C terminus via an AscI restriction site. TRPC3 with fluorescent proteins fused to the C terminus behaves indistinguishably from the native protein in both store-operated and second messenger-operated modes (12).2
Confocal MicroscopyFluorescence images were acquired with a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) using an argon laser and excitation at 488 nm through a 100x (oil immersion) objective lens.
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RESULTS |
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We designate the wild-type DT40 cells produced by these transfections T3low-WT and T3high-WT for cells transfected with 10 and 100 µg/ml, respectively, and T3low-KO and T3high-KO for IP3R-KO cells. Along with TRPC3, the cells were co-transfected with 50 µg/ml human M5 muscarinic receptor (in pcDNA3) and a construct encoding EYFP as a transfection marker (10 µg/ml). Co-transfection efficiency ranged between 6080% for EYFP positive (EYFP+) cells exhibiting carbachol-induced Ca2+ release. For those cells transfected with both M5 and TRPC3, co-transfection efficiency ranged between 7080% based on the number of EYFP+ cells exhibiting carbachol-induced Ba2+ entry (a functional marker of TRPC3 activation; see below). It is well established that increasing DNA copy number through the plasmid copy number leads to significant increases in protein levels (20). Control transfections run in parallel using either 50 or 100 µg/ml EYFP showed that increasing plasmid concentration did not enhance transfection efficiency (i.e. number of transfected cells) but markedly increased expression of the coded protein (EYFP-dependent cell fluorescence; not shown). Plasmid concentrations up to 200 µg/ml did not affect cell growth and/or viability. To confirm differential expression levels of TRPC3 in T3low and T3high DT40 cells, a construct with TRPC3 fused to the Topaz fluorescent protein (18) in pcDNA3 was used (T3T).3 Wild-type DT40 cells were transiently transfected with either 10 or 100 µg of T3T (designated T3Tlow-WT and T3Thigh-WT, respectively) or the equivalent amount of pcDNA3 carrying cDNA for Topaz protein alone (mock-transfected cells). Transfected cells were analyzed for T3T expression by confocal microscopy. In T3Thigh-WT cells, TRPC3 appeared to be localized in punctate spots in the plasma membrane (Fig. 1A). With microscope settings optimal for T3Thigh visualization (see legend to Fig. 1 for details), in T3Tlow-WT cells the plasma membrane labeling was barely detectable (Fig. 1B), consistent with the notion that lower TRPC3 expression levels are attained when lower amounts of plasmid are used. Cells transfected with equivalent amounts of pcDNA3-Topaz showed a fluorescence labeling confined exclusively to the cytosol (not shown).
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To evaluate a TRPC3-dependent cation entry under the different transfection conditions, either Ca2+ or Ba2+ entry was fluorometrically evaluated in single EYFP+ cells. We first assessed the influence of store depletion on cation entry in TRPC3-transfected cells. It is well known that, in DT40 cells, passive depletion of endogenous stores following blockade of the sarco-endoplasmic reticulum Ca2+/Mg2+-ATPases with thapsigargin results in activation of the CCE pathway (16, 21, 22). Unlike other expression systems (e.g. HEK293 cells), DT40 cells have a CCE poorly permeable to Ba2+ (14, 15). In our previous studies, we exploited this property of DT40 cells and reported that, when 10 mM Ba2+ was used, no store-operated entry was observed in non-transfected cells, whereas TRPC3 cells showed a robust Ba2+ entry (14, 19). However, in our more recent studies, we find that some untransfected DT40 cells do show a store-operated entry of 10 mM Ba2+. Thus, although it is still possible to document store-operated Ba2+ entry in quantitative terms (see, for example, Fig. 4A), we have now turned to a technique utilizing Ca2+ and specific channel-inhibiting drugs to document store-operated TRPC3 behavior. 2-Aminoethoxydiphenyl borane (2APB) is a reliable blocker of store-operated Ca2+ entry in most cells (see Ref. 23 and references therein). 2APB substantially inhibits endogenous CCE in DT40 cells but has a negligible effect on TRPC3 store-dependent activity in these cells (19); thus 2APB provides an alternative approach to monitor TRPC3-mediated Ca2+ entry without the contribution of the endogenous channels.
As shown previously for wild-type DT40 cells (16, 21, 22), mock-transfected cells respond to thapsigargin with a transient increase in cytosolic Ca2+ reflecting passive depletion of Ca2+ stores due to a blockade of endoplasmic reticulum Ca2+ pumps. Once Ca2+ levels returned to baseline, 30 µM 2APB was added, and 2 min later Ca2+ was added to the extracellular medium to evaluate activation of the store-operated pathway. CCE in mock-transfected wild-type cells was almost completely blocked (Fig. 2A), confirming the previously reported action of 2APB on the endogenous channels (19). However, wild-type DT40 cells transiently transfected with a low amount of TRPC3-coding vector (T3low-WT) showed a substantial, 2APB-insensitive Ca2+ entry after thapsigargin-induced store depletion (36 of 45 cells, Fig. 2A), consistent with our earlier report (19). Basal Ca2+ permeability was not affected by TRPC3 expression, as Ca2+ addition to T3low-WT cells not exposed to thapsigargin but maintained in Ca2+-free medium caused no detectable increase in cytosolic Ca2+ (not shown). 1 µM Gd3+ completely blocks store-operated TRPC3-mediated Ca2+ entry in response to thapsigargin in DT40 cells (19). As shown in Fig. 2A, once the peak of TRPC3-mediated Ca2+ entry was reached, the addition of 1 µM Gd3+ rapidly reversed this entry. However, applying the above described protocol to T3high-WT cells did not result in the appearance of store-operated Ca2+ entry (0 of 53 cells, Fig. 2B), which is in line with published evidence indicating that exogenously expressed TRPC3 usually results in a channel that is not sensitive to intracellular store depletion (Ref. 8 and references therein). Basal Ca2+ permeability was not affected by TRPC3 expression at higher levels (not shown), again indicating the absence of constitutive channel activity.
We hypothesized previously (14) that the expression of TRPC3 in DT40 might be lower than in, for example, HEK293 cells in part because of the relative inefficiency in avian cells of the constitutive cytomegalovirus (CMV) promoter in pcDNA3. In support of this contention, we found that the functional expression of the M5 muscarinic receptor was substantially improved when the gene was driven by an avian -actin promoter (
AP) than when driven by a CMV promoter2 (in the current study, we circumvent the problem with CMV by increasing the concentration of the M5 plasmid 5-fold compared with the amount necessary for the
AP construct; see Ref. 17, in which the
AP construct was used in the amount of 10 µg/ml). Thus, we subcloned the cDNA coding for TRPC3 into the expression vector lcf201 under control of the avian
AP. As for T3high-WT cells, wild-type DT40 cells transiently expressing
AP-driven TRPC3 (transfected with 10 µg/ml) did not show store-operated Ca2+ entry upon depletion of stores with thapsigargin (0 of 38 cells, Fig. 2B, gray trace). When cells were transfected with a combination of the CMV-driven and
AP-driven plasmids (10 µg/ml each), again no store-operated Ca2+ entry was observed (not shown). However, cells transfected with the
AP construct showed the expected carbachol-induced Ba2+ entry when the
AP-driven TRPC3 was co-transfected with the muscarinic M5 receptor (see below), which is consistent with a higher level of channel expression under control of the chicken promoter.
These results clearly show a disappearance of store-operated entry with more highly expressed TRPC3 (TRPC3high) in DT40 cells, whether by increasing plasmid concentration or by use of a more efficient promoter. We next examined the effect of differential expression levels on channel behavior when receptor-dependent activation of the PLC pathway occurs.
We showed earlier that, in DT40 cells, the activation of TRPC3low through agonist-induced activation of PLC activates TRPC3 through a store-depletion mechanism. A higher level of expression of TRPC3 in wild-type DT40 cells (T3high-WT) transiently expressing the M5 muscarinic receptor resulted in robust carbachol-induced Ba2+ entry (59 of 76 cells, Fig. 3A, black trace) that was not observed in cells transfected with the M5 receptor only (Fig. 3A, gray trace) or with TRPC3 only (not shown). In contrast to TRPC3low (14), cation permeation of TRPC3high could be seen with as little as 2 mM Ba2+; this may be due to the greater number of channels or possibly the greater relative Ba2+ permeability of the channels at high expression. Additionally, in clear contrast to the observations made for TRPC3low (Ref. 19 and Fig. 3B), Gd3+ (5 µM) did not appear to block agonist-induced Ca2+ entry in TRPC3high cells (Fig. 3B); in the presence of Gd3+, 0 of 41 TRPC3low cells responded, whereas 26 of 35 TRPC3high cells responded. This lack of sensitivity to Gd3+ is also observed when TRPC3 is exogenously expressed in other cell lines.
We next evaluated the behavior of TRPC3 when expressed in IP3R-KO DT40 cells. With low expression in IP3R-KO cells, PLC stimulation does not lead to activation of the channel, indicating an absolute requirement on IP3 receptor expression for agonist activation of TRPC3low, presumably because IP3 receptors are necessary for agonists to deplete Ca2+ stores (14). Consistent with this interpretation, carbachol stimulation of IP3R-KO cells transiently co-transfected with the M5 muscarinic receptor, the type-3 rat IP3 receptor, and TRPC3low resulted in a rapid release of calcium from stores followed by activation of TRPC3-mediated Ba2+ entry (10 mM Ba2+, 18 of 29 cells, Fig. 4A). Neither transient Ca2+ release nor Ba2+ entry was detected in IP3R-KO cells co-transfected only with M5 receptor and TRPC3low (0 of 33 cells responded, Fig. 4A). When IP3R-KO cells were transiently co-transfected with the M5 muscarinic receptor and TRPC3high, carbachol addition failed to induce release, but Ba2+ addition to the extracellular medium gave rise to a robust Ba2+ entry (60 of 81 cells, Fig. 4B) that was not observed in cells transfected with M5 receptor only (Fig. 4B). As for wild-type cells, T3high-KO cells lacking the muscarinic receptor did not show carbachol-induced Ba2+ entry (not shown). These results contrast sharply with those for TRPC3low expressing IP3R-KO cells.
It is known that members of the TRPC3/6/7 subfamily of TRPC channels can be activated by synthetic DAGs (13). We demonstrated previously that TRPC3low expression in either wild-type or IP3R-KO DT40 cells results in a channel sensitive to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant DAG (14). Thus, we examined the effect of OAG on TRPC3-mediated Ba2+ influx in both wild-type and IP3R-KO DT40 cells expressing high levels of TRPC3. In both mock- and TRPC3-transfected cells, OAG treatment did not affect either cytosolic Ca2+ levels or Ca2+ content of the stores (not shown). The addition of OAG to both T3high-WT and T3high-KO cells significantly stimulated Ba2+ influx (WT, 36 of 48 cells; KO, 31 of 41 cells, Fig. 5), whereas there was no effect on cation entry in mock-transfected cells. In both cell types, OAG-induced Ba2+ entry was comparable with that seen with receptor stimulation (Figs. 3 and 4) and was similar in T3high-WT and T3high-KO cells, indicating that, as for receptor-stimulation, OAG action is independent of the IP3R. A similar conclusion was drawn for TRPC3low (14), suggesting that OAG activation of TRPC3 involves a mechanism that is independent on the expression level of the channel, probably reflecting an intrinsic property of the TRPC3 protein itself.
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DISCUSSION |
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In the current study, we increased TRPC3 expression by two different strategies, i.e. by increasing the concentration of TRPC3-containing plasmid and by expressing TRPC3 under control of a more efficient promoter. The results clearly demonstrate that increasing the level of expression of TRPC3 in DT40 cells indeed results in the disappearance of store-operated behavior and the appearance of an IP3R-independent, receptor-activated behavior. The over-expression strategies were very efficient in causing this transformation of behavior; of 53 cells observed following transfection with the CMV promoter construct (100 µg/ml) and 38 cells observed following transfection with the -actin promoter construct (i.e. TRPC3high), not one cell exhibited store-operated entry (Fig. 2). Likewise, with a protocol that clearly reveals receptor-activated entry that is independent of store-depletion (i.e. in the absence of IP3 receptor, Fig. 4A), not one of 33 cells showed activation of entry following transfection with CMV-driven construct (i.e. TRPC3low) (10 µg/ml).
To our knowledge, this is the first demonstration of a channel protein functioning in two distinct ways depending on the expression level. The behavior observed at higher levels of expression confirms the findings of Venkatachalam et al. (15) and resolves this apparent contradiction in the literature. In addition, recent observations in our laboratory indicate that the expression of TRPC3 in DT40 cells, under low expression conditions, is also significantly lower than that in HEK293 cells wherein the channel is receptor activated.3 Yue et al. (26) similarly suggested that a diminished level of expression favors a store-operated mechanism for the TRPV6 channel. In this case, store-operated behavior was observed only at very short times after transfection, and, with more prolonged times, an unregulated channel resulted (26).
What might be the basis for this change in channel behavior as a function of expression level? It is perhaps not surprising to see new and/or unusual behavior of proteins as their expression level is artificially increased, but it is somewhat more difficult to understand why a particular characteristic, in this instance regulation by store depletion, is lost. One possibility is that TRPC3 channels do not function alone when in store-operated mode but must associate with other proteins in proper stoichiometric arrangements. Dramatically increasing one member of such a signaling complex may increase the likelihood of forming incomplete complexes simply by dilution of a limited pool of accessory proteins provided by the cell. What might these additional proteins or factors be? Functional TRP-based channels are apparently tetramers (7, 27). Endogenous TRP-encoded channels may form heterotetramers, whereas it is expected that heterologously overexpressed channels mainly give raise to homotetramers (14, 28). In the case of TRPC subfamily members, Hofmann et al. (27) demonstrated that TRPC3 can only form complexes with the closely related channels TRPC6 and/or TRPC7. However, interaction with proteins outside of the TRPC subfamily was not examined. In addition, other kinds of Ca2+ channels associate with subunits that are not necessarily a part of the pore-forming channels themselves (29). It is not known whether such subunits are involved in the formation of functional TRPC channels.
A final question is this. Which of the two modes of behavior represents the physiological function of TRPC3 channels? One might imagine that the behavior seen at the lowest expression level is likely to be the more physiologically relevant, but this need not necessarily be true. At the lower concentrations, TRPC3 may be imposing itself within a signaling structure into a role normally played by another somewhat related channel. There are reports in the literature of endogenous channels that could fit either behavior, e.g. store-operated non-selective cations channels (30), including the suggestion of a channel that can be regulated by both store-depletion and DAG (31), and receptor- and DAG-activated non-selective cation channels (32, 33). Thus, for the moment, we may consider the intriguing possibility that either or even both of these TRPC3 behaviors correspond to a physiological mode of TRPC3 channel regulation, depending on the specific cellular environment.
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FOOTNOTES |
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To whom correspondence should be addressed: NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail: Putney{at}niehs.nih.gov.
1 The abbreviations used are: PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3R-KO, IP3R knock-out; CCE, capacitative calcium entry; SOC, store-operated channel; TRP, transient receptor potential; TRPC canonical TRP; DAG, diacylglycerol; TRT, TRPC3-Topaz fusion protein; HEK293, human embryonic kidney 293; EYFP, enhanced yellow fluorescent protein; 2APB, 2-aminoethoxydiphenyl borane; WT, wild-type; CMV, cytomegalovirus; AP,
-actin promoter; OAG, 1-oleoyl-2-acetyl-sn-glycerol.
2 G. Vazquez, B. J. Wedel, M. Trebak, G. St. J. Bird, and J. W. Putney, Jr., unpublished observations.
3 Wedel, B. J., Vazquez, G., McKay, R. R., Bird, G. St. J., and Putney, J. W., Jr. (May 2, 2003) J. Biol. Chem. DOI 10.1074/jbc.M303890200.
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ACKNOWLEDGMENTS |
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REFERENCES |
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