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Expression Level of the Canonical Transient Receptor Potential 3 (TRPC3) Channel Determines Its Mechanism of Activation

2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês

10.1074/jbc.m302162200

ISSN

1083-351X

Autores

Guillermo Vázquez, Barbara J. Wedel, Mohamed Trebak, Gary S. Bird, James W. Putney,

Tópico(s)

Phytochemicals and Antioxidant Activities

Resumo

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian β-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression. Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian β-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression. In most nonexcitable cells, calcium signaling initiated through cell membrane receptors coupled to phospholipase C (PLC) 1The 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; βAP, β-actin promoter; OAG, 1-oleoyl-2-acetyl-sn-glycerol. results in production of inositol 1,4,5-trisphosphate (IP3) (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2272) Google Scholar). IP3 induces the release of Ca2+ from the endoplasmic reticulum and the subsequent influx of Ca2+ across the plasma membrane through the capacitative calcium entry (CCE) or the store-operated calcium entry pathway (3Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 4Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar, 5Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 6Barritt G.J. Biochem. J. 1999; 337: 153-169Crossref PubMed Scopus (281) Google Scholar). Although CCE has been widely studied in diverse cell types, the molecular identity of store-operated channels (SOCs) and the signal by which store emptying activates those channels remain uncertain. Mammalian homologues of the Drosophila transient receptor potential (TRP) channel have been proposed as candidates for SOCs (7Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 8Zitt C. Halaszovich C.R. Lückhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar). Among the members of the canonical TRP (TRPC) subfamily (designated TRPC1 through TRPC7), human TRPC3, first cloned by Zhu et al. (9Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar), has been shown in many heterologous expression systems to behave as a receptor-activated channel with constitutive activity that cannot be further increased by Ca2+ store depletion (10Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 11Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar, 12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar). Furthermore, Hofmann et al. (13Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-262Crossref PubMed Scopus (1268) Google Scholar) showed that TRPC3 and its structural relative TRPC6 can be activated by diacylglycerols (DAGs), providing a possible mechanism of activation of these channels by phospholipase C-linked receptors independent of IP3 and store depletion. We recently demonstrated that TRPC3 is regulated by store depletion when transiently expressed in DT40 chicken B-lymphocytes (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar), and we proposed TRPC3 as a candidate for store-operated, non-selective cation channels. However, Venkatachalam et al. (15Venkatachalam K. Ma H.-T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar), conflicts with the non-store-operated, receptor-regulated behavior reported by us for HEK293 cells (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar). We thus suggested that the mode of activation of TRPC3 channels may depend on the level of expression (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar). 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. Cell Culture, Transfection, and Measurement of Intracellular Calcium—The chicken B lymphocyte cell line DT40 and the mutant variant in which the genes for all three IP3R types were disrupted were obtained from the Institute of Physical and Chemical Research (RIKEN; RIKEN Cell Bank numbers RCB1464 and RCB1467). Cells were cultured essentially as described in Sugawara et al. (16Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). Loading of the cells with the Ca2+ indicator Fura-2 and the ratiometric measurement of intracellular Ca2+ were accomplished as described previously (17Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (55) Google Scholar). DT40 cells were transiently transfected by electroporation (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar) 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 β-actin promoter for expression in DT40 cells. Cells were assayed 17–25 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 (17Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (55) Google Scholar). Basal Ca2+ levels in both wild-type and IP3 receptor knock-out (IP3R-KO) (16Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar) DT40 cells were similar (around 110–135 nm). Under the conditions of measurement, EYFP expression did not contribute significant fluorescence. In Figs. 2, 3, 4, 5, average traces from 8–14 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.Fig. 3Activation of highly expressed TRPC3 (100 μg/ml) through the G-protein-coupled M5 muscarinic receptor (A) and the relative sensitivity of TRPC3 to Gd3+ with different degrees of expression (B).A, wild-type DT40 cells were co-transfected with the M5 receptor (solid gray line), 100 μg/ml TRPC3-coding pcDNA3 (solid black line), or the equivalent amount of vector alone (dotted line). Following loading with Fura-2, cells were incubated in nominally Ca2+-free medium and then exposed to 300 μm carbachol. Where indicated, Ba2+ (2 mm) was added to the medium. Representative traces from five independent experiments are shown. B, wild-type DT40 cells were co-transfected with the M5 receptor and either 10 μg/ml or 100 μg/ml (as indicated) TRPC3-coding pcDNA3. Cells were maintained in nominally Ca2+-free medium, exposed to 300 μm carbachol, and then Ca2+ (1.5 mm) was added where indicated. Gd3+ (5 μm) was present throughout the measurements. Representative traces from four independent experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Expression level of TRPC3 determines channel dependence upon IP3 receptor expression when activated through the G-protein-coupled M5 muscarinic receptor. Ba2+ influx was measured as an indicator of TRPC3 activity in Fura-2-loaded DT40 cells transiently expressing the human M5 muscarinic receptor. A, the M5 receptor was co-transfected into IP3R-KO DT40 cells with 10 μg/ml TRPC3-coding pcDNA3 and either the rat IP3R-3 or its vector (No IP3R). Cells were maintained in nominally Ca2+-free medium, exposed to 300 μm carbachol, and then Ba2+ (10 mm) was added where indicated. Traces are representative of four independent experiments. B, the M5 receptor was co-transfected into IP3R-KO DT40 cells with either 100 μg/ml TRPC3-coding pcDNA3 or its vector (M5 only). Cells were maintained in nominally Ca2+-free medium, exposed to 300 μm carbachol, and then Ba2+ (2 mm) was added where indicated. Shown are traces representative of five independent experimentsView Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5OAG-induced Ba2+ entry in DT40 cells expressing high levels of TRPC3. Ba2+ influx was measured in Fura-2-loaded wild type (A) or IP3R-KO (B) cells transiently transfected with either 100 μg/ml TRPC3-coding pcDNA3 or its vector (Mock), as indicated. The cells were maintained in a nominally Ca2+-free medium and then exposed to the membrane-permeant DAG analog OAG (200 μm) together with Ba2+ (2 mm). Data are representative of four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To generate the TRPC3-Topaz fusion construct (T3T), TRPC3 was fused to the brighter EYFP version Topaz (18Tsien R.Y. Ann. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar) 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 (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar). 2G. Vazquez, B. J. Wedel, M. Trebak, G. St. J. Bird, and J. W. Putney, Jr., unpublished observations. Confocal Microscopy—Fluorescence 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 100× (oil immersion) objective lens. Recently, we demonstrated that human TRPC3 is regulated by Ca2+ store depletion when transiently expressed in DT40 chicken B-lymphocytes (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar), whereas Venkatachalam et al. (15Venkatachalam K. Ma H.-T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) expressed TRPC3 in DT40 cells and obtained a receptor-activated but not store-operated channel. In addition, in our own laboratory, TRPC3 expressed in HEK293 cells was receptor-activated and not store-operated (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar). We speculated (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar) that the coupling mechanism for TRPC3 channels might depend on the level of expression. To address this hypothesis directly, we increased TRPC3 expression in DT40 cells by increasing 10-fold (100 μg/ml) the amount of plasmid carrying cDNA coding for TRPC3 compared with that used in our previous studies (10 μg/ml; as in Refs. 14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar and 19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), increasing significantly, presumably, the number of cDNA copies per transfected cell. For comparison, we also carried out parallel transfections with plasmid coding for TRPC3 (10 μg/ml). 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 60–80% for EYFP positive (EYFP+) cells exhibiting carbachol-induced Ca2+ release. For those cells transfected with both M5 and TRPC3, co-transfection efficiency ranged between 70–80% 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 (20Smolke C.D. Keasling J.D. Biotechnol. Bioeng. 2002; 78: 412-424Crossref PubMed Scopus (29) Google Scholar). 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 (18Tsien R.Y. Ann. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar) in pcDNA3 was used (T3T). 3Wedel, 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. 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). 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 (16Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar, 21Broad L.M. Braun F.-J. Lièvremont J.-P. Bird G.St.J. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22Ma H.-T. Venkatachalam K. Li H.-S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Unlike other expression systems (e.g. HEK293 cells), DT40 cells have a CCE poorly permeable to Ba2+ (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar, 15Venkatachalam K. Ma H.-T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar, 19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). 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. 23Bootman M.D. Collins T.J. Mackenzie L. Roderick H.J. Berridge M.J. Peppiatt C.M. FASEB J. 2002; 16: 1145-1150Crossref PubMed Scopus (611) Google Scholar and references therein). 2APB substantially inhibits endogenous CCE in DT40 cells but has a negligible effect on TRPC3 store-dependent activity in these cells (19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar); 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 (16Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar, 21Broad L.M. Braun F.-J. Lièvremont J.-P. Bird G.St.J. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22Ma H.-T. Venkatachalam K. Li H.-S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), 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 (19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). 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 (19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). 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 (19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). 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. 8Zitt C. Halaszovich C.R. Lückhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar) 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 β-actin promoter (βAP) 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 βAP construct; see Ref. 17Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (55) Google Scholar, in which the βAP 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 βAP. As for T3high-WT cells, wild-type DT40 cells transiently expressing βAP-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 βAP-driven plasmids (10 μg/ml each), again no store-operated Ca2+ entry was observed (not shown). However, cells transfected with the βAP construct showed the expected carbachol-induced Ba2+ entry when the βAP-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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar), 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. 19Trebak M. Bird G.St.J. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar). 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 (13Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-262Crossref PubMed Scopus (1268) Google Scholar). 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar). 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 (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar), 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. The human isoform of TRPC3, originally cloned by Zhu et al. (9Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar), is one of the most extensively characterized members of the TRPC subfamily of cation channels. Although initial studies indicated that the expression of TRPC3 in heterologous cell systems increased CCE (9Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar), it was shown later that this reflected a constitutive rather than regulated mode of Ca2+ entry (10Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). In fact, in most subsequent studies in which TRPC3 was exogenously expressed in cell lines, the channel behaved as a receptor-activated non-selective cation channel not dependent on store-depletion (11Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar, 12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar, 24Kamouchi M. Philipp S. Flockerzi V. Wissenbach U. Mamin A. Raeymaekers L. Eggermont J. Droogmans G. Nilius B. J. Physiol. (Lond.). 1999; 518: 345-358Crossref Scopus (163) Google Scholar). However, we recently demonstrated that store depletion does regulate TRPC3 when the channel is transiently expressed in DT40 chicken B-lymphocytes (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar). At about the same time, Venkatachalam et al. transiently expressed TRPC3 in both wild-type and IP3R-KO DT40 cells but observed a channel whose activation turned out to be strictly dependent upon receptor stimulation of the PLC pathway but was not regulated by store depletion (15Venkatachalam K. Ma H.-T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These authors proposed that, in DT40 cells, TRPC3 forms receptor-operated cation channels likely activated through PLC-dependent generation of DAG. Similarly, in our own laboratory we found that, in HEK293 cells, TRPC3 is not a store-operated channel (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. Bird G.St.J. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar) but appears to function as a DAG-regulated channel (25Trebak M. Bird G.St.J. McKay R.R. Birnbaumer L. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 16244-16252Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). We speculated that the store-operated behavior of TRPC3 in DT40 cells might result from a lower level of expression as compared with HEK293 cells (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar). 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 β-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. (15Venkatachalam K. Ma H.-T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) 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. (26Yue L. Peng J.-B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar) 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 (26Yue L. Peng J.-B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar). 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 (7Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (358) Google Scholar, 27Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar). Endogenous TRP-encoded channels may form heterotetramers, whereas it is expected that heterologously overexpressed channels mainly give raise to homotetramers (14Vazquez G. Lièvremont J.-P. Bird G.St.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (155) Google Scholar, 28Wu X. Babnigg G. Villereal M.L. Am. J. Physiol. 2000; 278: C526-C536Crossref PubMed Google Scholar). In the case of TRPC subfamily members, Hofmann et al. (27Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (634) Google Scholar) 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 (29Catterall W.A. Annu. Rev. Cell Dev. Biol. 2001; 16: 521-555Crossref Scopus (1958) Google Scholar). 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 (30Trepakova E.S. Gericke M. Hirakawa Y. Weisbrod R.M. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2001; 276: 7782-7790Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), including the suggestion of a channel that can be regulated by both store-depletion and DAG (31Su Z. Csutora P. Hunton D. Shoemaker R.L. Marchase R.B. Blalock J.E. Am. J. Physiol. 2001; 280 (-C1292): C1284Crossref PubMed Google Scholar), and receptor- and DAG-activated non-selective cation channels (32Tesfai Y. Brereton H.M. Barritt G.J. Biochem. J. 2001; 358: 717-726Crossref PubMed Scopus (87) Google Scholar, 33Jung S. Strotmann R. Schultz G. Plant T.D. Am. J. Physiol. 2002; 282: C347-C359Crossref PubMed Scopus (222) Google Scholar). 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. We are grateful to Drs. John O'Bryan and Carl Bortner for constructive comments and suggestions.

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