Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709
Submitted 19 July 2004 ; accepted in final form 13 August 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
calcium signaling; nonselective cation channels
Speculation regarding the molecular nature of store-operated channels arose from studies of signaling events and molecular components involved in phototransduction in Drosophila melanogaster. These investigations led to identification and cloning of the transient receptor potential (trp) gene (5, 21). The trp gene encodes for TRP protein, which is a component of the Ca2+-permeable, nonselective cation channels underlying sustained Ca2+ entry upon light activation of PLC (7, 18). Seven mammalian homologs with structural and functional similarities to Drosophila TRP have been identified and cloned (3, 2224, 35, 41, 43, 45, 47). They constitute the "classical" or "canonical" TRP family (designated TRPC1TRPC7), which is one family among the larger TRP superfamily of cation channels (2, 8, 10, 19, 20, 20, 28, 32, 40, 46). The TRPC family can be divided into four different subfamilies based on structural and functional characteristics: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5. TRPC4 and TRPC5 share 65% homology based on amino acid sequence, whereas members of the TRPC3/6/7 subfamily have even higher amino acid identity, 7080%, with TRPC3 and TRPC7 being slightly more similar to each other than to TRPC6. In many cell types, TRPC have been proposed to encode for components of native, nonselective cation channels activated by a mechanism dependent on receptor-mediated activation of PLC (20, 38, 46). Since their initial discovery, the activation mechanisms of members of the TRPC3/6/7 subfamily have been investigated in several different cell lines and using various experimental approaches, leading to conflicting evidence regarding their activation mechanism. For example, the majority of studies have concluded that TRPC3 is activated by agonist receptors linked to PLC, but not by store depletion. However, when transiently expressed in chicken B lymphocyte DT40 cells, TRPC3 was shown to behave as a store-operated channel (31, 36, 37). In this instance, it was found that a very low expression level of TRPC3, rather than a different cell environment, favored its behavior as a store-operated channel (37).
As has become almost commonplace in the TRPC field, the two studies reported to date, performed by two independent laboratories, regarding the mechanism of activation of TRPC7 have also led to conflicting conclusions. Okada et al. (23) first cloned TRPC7 and investigated its mode of activation. They transiently expressed the murine ortholog of TRPC7 in human embryonic kidney (HEK)-293 cells and reported that it behaved as a nonselective cation channel activated via DAG after receptor-mediated activation of PLC. However, they also reported that the channel was clearly not activated by store depletion. Subsequently, Riccio et al. (29) identified and cloned human TRPC7. They stably overexpressed the protein in HEK-293 cells and reported that TRPC7 behaves as a store-operated channel. However, their study included no assessment of TRPC7's constitutive activity (i.e., activity in the absence of apparent stimulation), which can sometimes create the appearance of regulation by store depletion (31, 32).2 In attempting to reconcile these discrepant findings in addition to the technical issues, two notable differences in the two studies must be considered. First, the sequence of TRPC7 cloned by Riccio et al. (29) was essentially identical to that obtained by Okada et al. (23), except that a leucine at position 111 (L111) was replaced by a proline (P111). Second, the study of Riccio et al. was performed by stably transfecting TRPC7 into HEK-293 cells, whereas Okada et al. used transient transfection. On the basis of previous experience with TRPC3 in our laboratory (37), we reasoned that subtle differences in structural or expression conditions might account for the apparent distinct gating mechanism of TRPC7.
Therefore, to reexamine the mode of activation of TRPC7, we stably as well as transiently transfected HEK-293 cells with either wild-type (wt)-TRPC7 (L111-TRPC7) or L111P-TRPC7. We then examined the ability of methacholine, an agonist known to stimulate PLC-linked muscarinic receptors, and thapsigargin, an intracellular Ca2+ store-depleting agent, to activate TRPC7 channels. Our findings demonstrate that TRPC7 can function as both a receptor- and a store-operated channel when ectopically expressed in HEK-293 cells. In this regard, TRPC7 differs from TRPC3 and TRPC6, which, in HEK-293 cells, can be activated only through PLC activation and not by passive store depletion. To our knowledge, this study provides the first demonstration that a channel protein can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories with regard to TRPC7 regulation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and transfection. HEK-293 cells stably expressing TRPC3-green fluorescent protein (GFP) fusion protein were described previously (17). Transient and stable transfections of wild-type HEK-293 cells (wt-HEK-293) obtained from American Type Culture Collection (Manassas, VA) were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Wt-HEK-293 cells were transfected with either pcDNA3 vector containing the coding sequence for human TRPC6 [kindly provided by Dr. T. Gudermann, Institut für Pharmakologie und Toxikologie, Fachbereich Medizin, Philipps-Universitat Marburg, Marburg, Germany (9)]. Other cells were transfected with pcDNA3.1() expression vector encoding for either wild-type human TRPC7 [wt-TRPC7 (leucine at position 111), jointly supplied by Christine Murphy and Adrian Wolstenholm of University of Bath, Bath, UK, and John Westwick of Novartis, Horsham, UK] or L111P-TRPC7 (proline at position 111; see below). To generate stable TRPC6- and TRPC7-expressing cells, the transfected cells were grown for 4 wk under continuous selection with 500 µg/ml of geneticin (Invitrogen, Carlsbad, CA) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine (complete DMEM) in a humidified 95% air-5% CO2 incubator. TRPC6- and TRPC7-transfected cell populations were then tested for the ability of methacholine, an activator of PLC-linked muscarinic receptors, to activate the channels in the presence of 10 µM Gd3+ (to isolate TRPC responses; see below) by Ca2+ measurements performed on single cells (see RESULTS). Between 80 and 90% of TRPC6- and TRPC7-transfected cells showed receptor-mediated Ca2+ entry insensitive to Gd3+, a percentage of responding cells not significantly different from that observed for the TRPC3-GFP-expressing cell population (data not shown; see Ref. 17). For transient expression, HEK-293 cells were transiently transfected with pcDNA3.1() vector containing the sequence encoding for either wt-TRPC7 or the point mutant L111P-TRPC7, in which leucine at position 111 was replaced by proline, along with pEYFP-C1 vector (Clontech, Palo Alto, CA) as a marker for transfection. Cells were assayed 1830 h posttransfection.
Mutagenesis. A point mutation [single base change at position 332 (T to C), leading to a proline instead of a leucine at position 111] was introduced in the wt-TRPC7 coding sequence cloned into pcDNA3.1() vector using specific oligonucleotides and a single site-directed mutagenesis kit (Qiagen, Valencia, CA). The presence of the mutated nucleotide was verified by performing DNA sequencing.
Measurement of intracellular Ca2+. For wild-type and stable TRPC3-, TRPC6-, and TRPC7-expressing HEK-293 cells and transient wt-TRPC7- and L111P-TRPC7-expressing HEK-293 cells, Ca2+ and Ba2+ measurements were performed on single cells attached to coverslips. The coverslips were mounted in a Teflon chamber and incubated at 37°C for 30 min in complete DMEM containing 2 µM fura-2 AM (Molecular Probes, Eugene, OR). Cells were then washed and bathed in a nominally Ca2+-free HEPES-buffered saline solution (HBSS) for at least 10 min before Ca2+ and Ba2+ measurements were performed. The composition of the nominally Ca2+-free HBSS was (in mM) 140 NaCl, 4.7 KCl, 2 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, adjusted with NaOH. In some experiments, the NaCl of the HBSS was substituted with an equimolar quantity of KCl. To detect divalent cation entry, 2 mM Ca2+ or Ba2+ was added to the medium. For experiments performed under depolarizing conditions, the Ba2+ concentration was raised to 5 mM and then to 10 mM. For experiments in which cells were transiently transfected with TRPC, 4050 single cells were selected on the basis of enhanced yellow fluorescent protein (EYFP) expression, with fluorescence detected when excited at 488 nm and emission wavelength observed at 520 nm. In transiently and stably transfected cells, measurements of intracellular Ca2+ concentration ([Ca2+]i) changes as well as Ba2+ measurements with fura-2 were recorded and analyzed with a digital fluorescence imaging system (InCyt Im2; Intracellular Imaging, Cincinnati, OH) as described previously (30). For each experiment, with the exception of the one shown in Fig. 7, background fluorescence values at 340- and 380-nm excitation were obtained at the end of the experiment by addition of 4 µM ionomycin plus 20 mM Mn2+. These values were subtracted from individual values at each wavelength and at each time point of the experiment, and resulting background-corrected values were used to calculate fluorescence intensity ratios due to excitation at 340 and 380 nm. Because the signals in some instances derived from a mixture of Ca2+ and Ba2+, these ratio values were not processed further to obtain estimates of [Ca2+]i. All experiments were conducted at room temperature.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
TRPC3, TRPC6, and TRPC7 form channels activated by receptor-regulated PLC. As demonstrated by Trebak et al. (31), when Ca2+ is used to assess channel activation, misleading results can sometimes be obtained. For example, the inability of the endoplasmic reticulum to buffer Ca2+ entering through the channels (due to irreversible block of sarcoendoplasmic reticulum Ca2+-ATPase produced by thapsigargin) can exaggerate constitutive Ca2+ entry occurring through ion channels expressed in living cells, leading to the erroneous conclusion that these channels are activated upon store depletion. Likely this explains the small apparent store-operated Ca2+ entry in TRPC3-expressing HEK-293 cells reported by Zhu et al. (44) and by Kwan et al. (14) and could contribute to the apparent store-operated behavior of TRPC7 reported by Riccio et al. (29). To examine more quantitatively the mode of activation of TRPC3, TRPC6, and TRPC7, and to avoid complications caused by altered Ca2+ buffering, we used Ba2+ as a surrogate for Ca2+ (4, 13, 31, 33, 34). Furthermore, Ba2+ measurements were performed in the presence of 10 µM Gd3+, a concentration previously demonstrated to completely block endogenous store depletion-induced Ba2+ entry in wt-HEK-293 cells (15, 31), but not thought to block any other known Ca2+ permeable channel (27), including TRPC channels (Ref. 30 and the present study). The presence of Gd3+ ensured that any cation entry occurring in TRPC3-, TRPC6-, and TRPC7-expressing HEK-293 cells upon store depletion could be attributed only to expression of TRPC3, TRPC6, or TRPC7 and not to endogenous receptor-regulated or store-operated Ba2+ entry. As summarized in Fig. 1, cells expressing TRPC3 and TRPC7 showed similar constitutive Ba2+ entry in the presence of 10 µM Gd3+. However, no significant difference was observed between wt-HEK-293 and TRPC6-transfected HEK-293 cells. The substantial constitutive activities of TRPC3 and TRPC7 and the lesser constitutive activity of TRPC6 are consistent with previous findings (6, 17, 23, 31, 44). In the presence of 10 µM Gd3+, addition of 300 µM methacholine significantly activated TRPC3, TRPC6, and TRPC7 when expressed in HEK-293 cells, while wild-type cells showed no detectable Ba2+ entry in response to the same concentration of methacholine (31). Thus the fluorescence data indicate that TRPC3, TRPC6, and TRPC7 proteins form cation channels activated by PLC-coupled agonist, results consistent with those of previous reports (9, 11, 23).
|
|
|
|
|
|
The results discussed to this point indicate that when stably expressed in HEK-293 cells, TRPC7 forms both store-operated and receptor-operated channels. This finding, to our knowledge, is without precedent. The question then arises as to whether there exist separate populations of channels subject to these distinct modes of regulation. The results summarized in Fig. 8 indicate that this is unlikely to be the case. In the experiment shown in Fig. 8, TRPC7 channels were first activated by a maximal concentration of the synthetic analog of DAG, 1-oleoyl-2-acetyl-sn-glycerol (OAG). Subsequently, methacholine was added, which, in addition to activating PLC, would also cause depletion of Ca2+ stores. However, this depletion of Ca2+ stores did not appear to increase the steady-state Ca2+ entry. Thus, once the channels had been activated by OAG, there did not appear to be an additional population of channels that could then be activated by store depletion. Note that the results shown in Fig. 5 indicate that activation by PLC and DAG caused a greater Ca2+ entry than that seen with store depletion. This could mean either that there are some channels activated by DAG that are not coupled to store depletion or that all channels can be activated by either means, but to a greater extent by DAG.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Close examination of the two previous studies reveals two fundamental differences. First, the clone obtained by Riccio et al. carries a single base change compared with other clones from human (this study) and other species, resulting in a proline at position 111 instead of a leucine. Riccio et al. (29) speculated that this single amino acid substitution might be responsible for producing the store-operated phenotype. A second difference is that Riccio et al. used stably transfected HEK-293 cells, while Okada et al. (23) used transient transfection. Our findings indicate that it is the latter that likely explains the different results. We found no difference in the behavior of TRPC7 proteins with proline or leucine at position 111. Both were clearly store operated when stably expressed but only agonist activated when transiently expressed. Thus the current study reconciles the previous conflicting conclusions regarding the activation mechanism of TRPC7 (23, 29).
To our knowledge, this report describes the first demonstration of a channel protein being regulated by both receptor- and store-operated modes in the same cells. What might be the basis for a dual activation mechanism of a channel? In nonexcitable cells, receptor-mediated activation of PLC results not only in the production of both IP3 and DAG but also in the depletion of the intracellular stores. While TRPC3 and TRPC6 activation has been shown to be dependent strictly on DAG formed as a result of PLC activation and not on store depletion (9, 12, 17, 31, 39, 42, 44), the data presented in this study suggest that either store depletion or DAG production can activate TRPC7 when stably expressed in HEK-293 cells. In TRPC7-expressing cells, receptor-mediated activation of PLC resulted in a greater activation of TRPC7 than when activated by passive store depletion using thapsigargin (Fig. 3). However, agonist activation, which involves depletion of Ca2+ stores, did not increase entry beyond that obtained with OAG (Fig. 8), an agent that activates by acting as a DAG but without depleting stores. This would indicate that there are not separate populations of TRPC7 channels activated by different mechanisms, but rather a single population of channels that can be activated by store depletion or to a greater extent by DAG.
Taking into account the high degree of amino acid identity among members of the TRPC3/6/7 subfamily, one might speculate that this difference in sensitivity to store depletion presumably reflects subtle functional differences among the amino acid sequences of TRPC3, TRPC6, and TRPC7. Such differences may permit TRPC7 to associate with other proteins in proper stoichiometric arrangements to function as a store-operated channel when stably expressed in HEK-293 cells.
This report clearly demonstrates that in the HEK-293 cell environment, both wt-TRPC7 and L111P-TRPC7 were activated by store depletion when stably expressed in these cells, while when the two proteins were transiently expressed in the same HEK-293 cell environment, neither wt-TRPC7 nor L111P-TRPC7 behaved as store-operated channels. This suggests that the mode of expression can influence the behavior of a channel protein. Such a conclusion is not without precedent. For example, the majority of studies of TRPC3 have concluded that TRPC3 is activated by agonist receptors linked to PLC but not by store depletion in HEK-293 cells (16, 17, 30, 31, 44). Nevertheless, in a recent report, Vazquez et al. (37) demonstrated that when expressed in the avian B-cell line DT40, TRPC3 could behave as either a store-operated channel or a PLC-activated channel, depending on the level of expression. Although we have not investigated the levels of TRPC7 expression under these two transfection conditions, experience with other TRPC indicates that with the powerful cytomegalovirus promoter used, expression levels are generally similar. In addition, there are a number of important differences between the current findings with TRPC7 and earlier findings with TRPC3 (37). First, at low expression levels, TRPC3 produced only store-operated channels, and at high expression levels, it led to only DAG-activated channels, while for TRPC7 in the stable cells, both behaviors were observed. Also, the store-operated TRPC3 channels were blocked by Gd3+, while for TRPC7, clearly they are not. Thus we speculate that for TRPC7, stable transfection permits slow upregulation of other cellular components necessary to couple TRPC7 to store depletion. It is intriguing that the structurally similar TRPC3 does not show this phenomenon, and a comparison of changes in gene expression in cells stably expressing these two proteins may provide clues to the activation mechanism of store-operated channels in general.
In conclusion, the present results show that in HEK-293 cells, TRPC7 is activated both by receptor-mediated activation of PLC upon agonist stimulation and by store depletion. This behavior contrasts with that of TRPC3 and TRPC6, which, in our hands, formed only receptor-activated channels in this environment. These findings emphasize the importance of the mode of expression of channel proteins in studies aimed at elucidating their activation mechanism.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
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.
1 Although PLC activation inevitably leads to activation of store-operated channels, in this report when we refer to PLC-activated channels, we mean those channels that are specifically activated by PLC independently of store depletion.
2 This can occur when endoplasmic reticulum buffering is compromised, leading to an amplification of the cytoplasmic Ca2+ signal through constitutively open channels. The problem is alleviated by using a nontransported cation, such as Ba2+.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Birnbaumer L, Zhu X, Jiang M, Boulay G, Peyton M, Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, and Birnbaumer M. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc Natl Acad Sci USA 93: 1519515202, 1996.
3. Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E, and Birnbaumer L. Cloning and expression of a novel mammalian homolog of Drosophila Transient Receptor Potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 272: 2967229680, 1997.
4. Byron KL and Taylor CW. Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells. J Physiol 485: 455468, 1995.[Abstract]
5. Cosens DJ and Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature 224: 285287, 1969.[ISI][Medline]
6. Dietrich A, Schnitzler M, Emmel J, Kalwa H, Hofmann T, and Gudermann T. N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity. J Biol Chem 278: 4784247852, 2003.
7. Hardie RC and Minke B. Novel Ca2+ channels underlying transduction in Drosophila photoreceptors: implications for phosphoinositide-mediated Ca2+ mobilization. Trends Neurosci 16: 371376, 1993.[CrossRef][ISI][Medline]
8. Harteneck C, Plant TD, and Schultz G. From worm to man: three subfamilies of TRP channels. Trends Neurosci 23: 159166, 2000.[CrossRef][ISI][Medline]
9. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, and Schultz G. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397: 259262, 1999.[CrossRef][ISI][Medline]
10. Hofmann T, Schaefer M, Schultz G, and Gudermann T. Transient receptor potential channels as molecular substrates of receptor-mediated cation entry. J Mol Med 78: 1425, 2000.[CrossRef][ISI][Medline]
11. Hurst RS, Zhu X, Boulay G, Birnbaumer L, and Stefani E. Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK-293 cells. FEBS Lett 422: 333338, 1998.[CrossRef][ISI][Medline]
12. Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, and Mori Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular 1-adrenoceptor-activated Ca2+-permeable cation channel. Circ Res 88: 325332, 2001.
13. Kwan CY and Putney JW Jr. Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells: dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool. J Biol Chem 265: 678684, 1990.
14. Kwan HY, Huang Y, and Yao X. Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G. Proc Natl Acad Sci USA 101: 26252630, 2004.
15. Luo D, Broad LM, Bird GSJ, and Putney JW Jr. Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK-293 cells. J Biol Chem 276: 56135621, 2001.
16. Ma HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K, and Gill DL. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287: 16471651, 2000.
17. McKay RR, Szmeczek-Seay CL, Lièvremont JP, Bird GSJ, Zitt C, Jüngling E, Lückhoff A, and Putney JW Jr. Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3. Biochem J 351: 735746, 2000.[CrossRef][ISI][Medline]
18. Montell C. New light on TRP and TRPL. Mol Pharmacol 52: 755763, 1997.
19. Montell C. Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci STKE 2001: RE1, 2001. doi:10.1126/stke.2001.90.re1.
20. Montell C, Birnbaumer L, and Flockerzi V. The TRP channels, a remarkably functional family. Cell 108: 595598, 2002.[ISI][Medline]
21. Montell C and Rubin GM. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2: 13131323, 1989.[ISI][Medline]
22. Okada T, Shimizu S, Wakamori M, Maeda A, Kurosaki T, Takada N, Imoto K, and Mori Y. Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain. J Biol Chem 273: 1027910287, 1998.
23. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S, Ikenaka K, Imoto K, and Mori Y. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7: Ca2+-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor. J Biol Chem 274: 2735927370, 1999.
24. Philipp S, Cavalié A, Freichel M, Wissenbach U, Zimmer S, Trost C, Marguart A, Murakami M, and Flockerzi V. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J 15: 61666171, 1996.[Abstract]
25. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 112, 1986.[ISI][Medline]
26. Putney JW Jr. Capacitative calcium entry revisited. Cell Calcium 11: 611624, 1990.[ISI][Medline]
27. Putney JW Jr. Pharmacology of capacitative calcium entry. Mol Interventions 1: 8494, 2001.
28. Putney JW Jr and McKay RR. Capacitative calcium entry channels. Bioessays 21: 3846, 1999.[CrossRef][ISI][Medline]
29. Riccio A, Mattei C, Kelsell RE, Medhurst AD, Calver AR, Randall AD, Davis JB, Benham CD, and Pangalos MN. Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx. J Biol Chem 277: 1230212309, 2002.
30. Trebak M, Bird GSJ, McKay RR, Birnbaumer L, and Putney JW Jr. Signaling mechanism for receptor-activated TRPC3 channels. J Biol Chem 278: 1624416252, 2003.
31. Trebak M, Bird GSJ, McKay RR, and Putney JW Jr. Comparison of human TRPC3 channels in receptor-activated and store-operated modes: differential sensitivity to channel blockers suggests fundamental differences in channel composition. J Biol Chem 277: 2161721623, 2002.
32. Trebak M, Vazquez G, Bird GSJ, and Putney JW Jr. The TRPC3/6/7 subfamily of cation channels. Cell Calcium 33: 451461, 2003.[CrossRef][ISI][Medline]
33. Uvelius B, Sigurdson SB, and Johansson B. Strontium and barium as substitutes for calcium on electrical and mechanical activity in rat portal vein. Blood Vessels 11: 245259, 1974.[ISI][Medline]
34. Vanderkooi JM and Martonosi A. Sarcoplasmic reticulum: XII. The interaction of 8-anilino-1-naphthalene sulfonate with skeletal muscle microsomes. Arch Biochem Biophys 144: 8798, 1971.[ISI][Medline]
35. Vannier B, Peyton M, Boulay G, Brown D, Qin N, Jiang M, Zhu X, and Birnbaumer L. Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ channel. Proc Natl Acad Sci USA 96: 20602064, 1999.
36. Vazquez G, Lièvremont JP, Bird GSJ, and Putney JW Jr. Trp3 forms both inositol trisphosphate receptor-dependent and independent store-operated cation channels in DT40 avian B-lymphocytes. Proc Natl Acad Sci USA 98: 1177711782, 2001.
37. Vazquez G, Wedel BJ, Trebak M, Bird GSJ, and Putney JW Jr. Expression level of TRPC3 channel determines its mechanism of activation. J Biol Chem 278: 2164921654, 2003.
38. Venkatachalam K, van Rossum DB, Patterson RL, Ma HT, and Gill DL. The cellular and molecular basis of store-operated calcium entry. Nat Cell Biol 4: E263E272, 2002.[CrossRef][ISI][Medline]
39. Venkatachalam K, Zheng F, and Gill DL. Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278: 2903129040, 2003.
40. Vennekens R, Voets T, Bindels JM, Droogmans G, and Nilius B. Current understanding of mammalian TRP homologues. Cell Calcium 31: 253264, 2002.[CrossRef][ISI][Medline]
41. Wes PD, Chevesich J, Jeromin A, Rosenberg C, Stetten G, and Montell C. TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci USA 92: 96529656, 1995.[Abstract]
42. Zhang L and Saffen D. Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. J Biol Chem 276: 1333113339, 2001.
43. Zhu X, Chu PB, Peyton M, and Birnbaumer L. Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett 373: 193198, 1995.[CrossRef][ISI][Medline]
44. Zhu X, Jiang M, and Birnbaumer L. Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells: evidence for a non-capacitative calcium entry. J Biol Chem 273: 133142, 1998.
45. Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E, and Birnbaumer L. trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85: 661671, 1996.[ISI][Medline]
46. Zitt C, Halaszovich CR, and Lückhoff A. The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry. Prog Neurobiol 66: 243264, 2002.[CrossRef][ISI][Medline]
47. Zitt C, Zobel A, Obukhov AG, Harteneck C, Kalkbrenner F, Lückhoff A, and Schultz G. Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion. Neuron 16: 11891196, 1996.[ISI][Medline]