The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels
2005; Elsevier BV; Volume: 280; Issue: 42 Linguagem: Inglês
10.1074/jbc.m507606200
ISSN1083-351X
AutoresJean-Philippe Lièvremont, Takuro Numaga, Guillermo Vázquez, Loïc Lemonnier, Yuji Hara, Emiko Mori, Mohamed Trebak, Stephen E. Moss, Gary S. Bird, Yasuo Mori, James W. Putney,
Tópico(s)Herbal Medicine Research Studies
ResumoPhospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current Icrac was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPγS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels. Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current Icrac was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPγS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels. Transient receptor potential channels (TRPCs) 4The abbreviations used are: TRPCtransient receptor potential channelGTPγSguanosine 5′-3-O-(thio)triphosphateOAGoleyl acetyl glycerolBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidFura-2Fura-2-acetoxymethylesterIP3inositol 1,4,5-trisphosphatePAR2protease-activated receptor 2WTwild-type/cracCa2+ release-activated Ca2+ current. 4The abbreviations used are: TRPCtransient receptor potential channelGTPγSguanosine 5′-3-O-(thio)triphosphateOAGoleyl acetyl glycerolBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidFura-2Fura-2-acetoxymethylesterIP3inositol 1,4,5-trisphosphatePAR2protease-activated receptor 2WTwild-type/cracCa2+ release-activated Ca2+ current. are believed to function as multifunctional calcium-permeable cation channels (1Putney Jr., J.W. Trends Cell Biol. 2004; 14: 282-286Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Depending on cell type, expression level, or expression environment, the channels can be activated through the phospholipase C pathway, or in some instances, by depletion of intracellular Ca2+ stores (1Putney Jr., J.W. Trends Cell Biol. 2004; 14: 282-286Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 2Vazquez G. Wedel B.J. Aziz O. Trebak M. Putney Jr., J.W. Biochim. Biophys. Acta. 2004; 1742: 21-36Crossref PubMed Scopus (271) Google Scholar, 3Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.-T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (335) Google Scholar). The calcium-permeable cation channel, canonical transient receptor potential 7 (TRPC7) was originally shown to function as a phospholipase C-regulated channel, presumably through production of diacylglycerol (4Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). However, a subsequent report has provided evidence that TRPC7 could function as a store-operated channel (5Riccio A. Mattei C. Kelsell R.E. Medhurst A.D. Calver A.R. Randall A.D. Davis J.B. Benham C.D. Pangalos M.N. J. Biol. Chem. 2002; 277: 12302-12309Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Finally, it has been demonstrated that the different findings from the two laboratories likely result from differences in expression conditions. When TRPC7 is transiently expressed in HEK293 cells, it behaves as a diacylglycerol-activated channel; but when expressed stably, it appears to function as a capacitative or store-operated calcium entry channel (6Lievremont J.P. Bird G.S. Putney Jr., J.W. Am. J. Physiol. 2004; 287: C1709-C1716Crossref PubMed Scopus (69) Google Scholar). It is thus important to determine which, if either, of these two behaviors corresponds to the physiological function of TRPC7 when expressed endogenously. To date, there have been no studies of the physiological function of endogenously expressed TRPC7. Thus, in the current work, we utilized targeted homologous recombination to knock out TRPC7 in B-lymphocytes of DT40, a cell line previously shown to endogenously express both store-operated and non-store-operated pathways (7Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar, 8Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (54) Google Scholar, 9Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.-G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Although knock out of TRPC7 from DT40 cells caused significant alterations in cellular Ca2+ homeostasis and signaling, it did not result in a diminution in whole-cell Icrac current. Rather, in the avian B-cell line, TRPC7 appears to function as a phospholipase C-regulated, diacylglycerol-activated channel. transient receptor potential channel guanosine 5′-3-O-(thio)triphosphate oleyl acetyl glycerol 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid Fura-2-acetoxymethylester inositol 1,4,5-trisphosphate protease-activated receptor 2 wild-type Ca2+ release-activated Ca2+ current. transient receptor potential channel guanosine 5′-3-O-(thio)triphosphate oleyl acetyl glycerol 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid Fura-2-acetoxymethylester inositol 1,4,5-trisphosphate protease-activated receptor 2 wild-type Ca2+ release-activated Ca2+ current. Reagents—Thapsigargin, methacholine, and oleyl acetyl glycerol (OAG) were purchased from Calbiochem, San Diego, CA. BAPTA was purchased from Calbiochem (La Jolla, CA). Methacholine was purchased from Sigma. Fura-2-acetoxymethylester (Fura-2) was from Molecular Probes, Inc. Ca2+ and Ba2+ Measurements—The immortalized chicken B-lymphocyte cell line DT40 (RIKEN (Institute of Physical and Chemical Research) Cell Bank number RCB1464) and a mutant version with the gene for TRPC7 disrupted (see below) were maintained in suspension culture and loaded with the Ca2+ indicator Fura-2, as previously described (10Broad 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 (197) Google Scholar). The cells were then washed and bathed in Hepes-buffered physiological saline solution at room temperature at least 20 min before Ca2+ or Ba2+ measurements were made. In some experiments, a nominally Ca2+-free medium was used, which was identical in composition, except for the omission of added CaCl2. For all cell lines, measurements of Fura-2 fluorescence ratios, indicative of changes in intracellular Ca2+ or Ba2+, were recorded and analyzed with a digital fluorescence imaging system (InCyt Im2, Intracellular Imaging Inc., Cincinnati, OH), as described previously (11Trebak 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 (143) Google Scholar). All experiments were conducted at room temperature, and data were reported as the ratio of fluorescence due to excitation at 340 and 380 nm. Transmembrane divalent cation flux, either constitutive or stimulated, was estimated by measuring the initial rate of Ba2+ entry following the addition of 10 mm Ba2+ in fluorescence ratio units/min. Electrophysiology—Macroscopic membrane ion currents were recorded using the patch clamp technique in its whole-cell configuration. The currents were acquired using pCLAMP-9.2 (Axon Instruments, Foster City, CA) and analyzed off-line using Origin 6 (Microcal) software. The extracellular solution (osmolarity 310 mosmol/liter) contained (in mm): NaCl, 145; KCl, 5; Hepes, 10; MgCl2, 1; CaCl2, 2 or 10, as indicated; pH 7.3 (adjusted with NaOH). The intracellular pipette solution (osmolarity 290 mosmol/liter) contained (in mm): cesium-methanesulfonate, 145; BAPTA, 10; Hepes, 10; MgCl2, 1; CaCl2, 0 or 2.2; pH 7.2 (adjusted with CsOH). Patch pipettes were fabricated from borosilicate glass capillaries (WPI Instruments). The resistance of the pipettes varied between 3 and 5 mΩ. Necessary supplements were added directly to the respective solutions in concentrations that would not significantly change the osmolarity. Changes in the external solutions were carried out using a multibarrel puffing micropipette with common outflow that was positioned in close proximity to the cell under investigation. During the experiment, the cell was continuously superfused with the solution via puffing pipette to reduce possible artifacts related to the switch from static to moving solution and vise versa. Generation of TRPC7-deficient DT40 Cells—The strategy is summarized in Fig. 1. The chicken genomic TRPC7 DNA was obtained by PCR using pairs of primers: chTRPC7-P1, sense, 5′-TTGAAGTTTGTAGCACACGCAGTTTC-3′ and chTRPC7-P1FR, antisense, 5′-ACGACGCCACAAAGATGGACA-3′; chTRPC7-P1F sense, 5′-TGTCCATCTTTGTGGCGTCGT-3′ and chTRPC7-P2, antisense, 5′-GGTGTTGTTTATCATGGCTATTAGC, respectively. The targeting vector of TRPC7 was constructed by replacing the genomic sequence, which encodes the hydrophobic segment H-3–H-6 of chicken TRPC7, with a histidinol (hisD) or neomycin resistance gene (neo) cassette (12Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (586) Google Scholar). The upstream 4.3-kb and downstream 1.6-kb genomic sequences of TRPC7 were used as a targeting vector. DT40 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum, penicillin, streptomycin, and glutamine. The targeting vector was linearized and transfected sequentially into DT40 cells by electroporation (550 V, 25 microfarads). After isolation of several clones in the presence of 1 mg/ml histidinol or 2 mg/ml G418, genomic DNAs were prepared and analyzed by Southern blot analysis using the 3′-flanking probe (Fig. 1). Northern Blot Analysis—RNA blot hybridization analysis was carried out using total RNA (30 μg) from wild-type or TRPC7-/- cells. The probe used to detect TRPC7 RNA was amplified by reverse transcription-PCR using specific primers (chTRPC7-P1F and chTRPC7-P6, 5′-GATTACTCCATAGAGTACGTATCC-3′). The Random Primer DNA labeling kit, version 2 (Takara Shuzo) was used to 32P label the probe. Hybridization was performed at 42 °C in 50% formamide, 5× SSC, 50 mm sodium phosphate buffer (pH 7.0), 0.1% SDS, 5× Denhardt's solution, and 0.2 mg/ml sonicated herring sperm DNA, as described previously (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar). Transient Transfections—Transient transfections of the wild-type and TRPC7-/- DT40 cells were carried out by electroporation essentially as described previously (14Vazquez G. Wedel B.J. Trebak M. Bird G.St.J. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 21649-21654Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), with either the human isoform of TRPC7 (10 μg/ml) into pcDNA3 vector or vector alone (pcDNA3, mock transfected cells) along with enhanced yellow fluorescent protein (5 μg) as a marker for transfection. Cells were used for real-time fluorescence measurements 20–25 h post-transfection. In figures involving transient transfections, average traces from 6–10 enhanced yellow fluorescent protein-positive cells are shown for a single experiment, and these are representative of three independent experiments. Statistics—When comparisons were made between two groups, we used Student's t test; when three groups were analyzed, we used analysis of variance with Tukey-Kramer multiple comparisons as post hoc tests. In some instances, in which multiple knock-out cells gave (in every cell) no response at all, we simply provided the mean and S.E. of the wild-type cells and cited the number of knock-out cells that failed to respond. Knock Out of TRPC7 in DT40 B-lymphocytes Alters Thapsigargin-activated [Ca2+]i Signals—Initially, we sought to determine whether TRPC7 might play a role in either store-operated or non-store-operated calcium entry in a cell in which both modes of entry occur. We chose the avian pre-B-cell line DT40, because these cells have been shown to express the archetypical store-operated current, Icrac (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar, 15Prakriya M. Lewis R.S. J. Physiol. (Lond.). 2001; 536: 3-19Crossref Scopus (423) Google Scholar, 16Kiselyov K. Shin D.M. Shcheynikov N. Kurosaki M. Muallem S. Biochem. J. 2001; 360: 17-22Crossref PubMed Scopus (56) Google Scholar), as well as a less well characterized non-store-operated pathway (8Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (54) Google Scholar, 9Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.-G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and because of the relative ease of targeting genes in these cells for disruption by homologous recombination (7Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar, 17Buerstedde J.-M. Takeda S. Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 18Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar). Two independent DT40 cell lines null for the TRPC7 message (Fig. 1) were generated, as described under "Materials and Methods." The lines were designated TRPC7-/- clone 14.3 and TRPC7-/- clone 14.304. Fig. 2 shows initial experiments examining store-operated or capacitative calcium entry in wild-type and TRPC7-/- cells. Both knock-out lines differed from wild-type cells in two ways. First, both lines appeared to have substantially increased intracellular Ca2+ stores, based on the size of the response to thapsigargin in the absence of extracellular Ca2+. Similar results were obtained using the Ca2+ ionophore ionomycin to discharge the stores (not shown). Second, the rate of Ba2+ entry in response to store depletion by thapsigargin (a measure of store-operated divalent cation entry, (6Lievremont J.P. Bird G.S. Putney Jr., J.W. Am. J. Physiol. 2004; 287: C1709-C1716Crossref PubMed Scopus (69) Google Scholar, 19Kwan C.Y. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 678-684Abstract Full Text PDF PubMed Google Scholar, 20Byron K.L. Taylor C.W. J. Physiol. (Lond.). 1995; 485: 455-468Crossref Scopus (76) Google Scholar, 21Broad L.M. Powis D.A. Taylor C.W. Biochem. J. 1996; 316: 759-764Crossref PubMed Scopus (18) Google Scholar)) appeared to be significantly depressed in both lines (wild type, 0.063 + 0.009 ratio units/min, n = 193; clone 14.3, 0.009 + 0.002 ratio units/min, n = 205; clone 14.304, 0.023 + 0.004 ratio units/min, n = 180; analysis of variance, p < 0.0001; wild type versus clone 14.3, p < 0.001; wild type versus clone 14.304, p < 0.001; clone 14.3 versus clone 14.304, p > 0.05). The diminished rate of Ba2+ entry seen in the thapsigargin-treated TRPC7-/- cells is indicative of a diminished store-operated entry and thus might imply a role for TRPC7 in this pathway. However, we also considered the possibility that the enlarged thapsigargin-sensitive Ca2+ pool might retain Ca2+ for a longer time, resulting in less pool depletion and thus less activation of Ba2+ entry (see, for example, Ref. 22Leissring M.A. Akbari Y. Fanger C.M. Cahalan M.D. Mattson M.P. LaFerla F.M. J. Cell Biol. 2000; 149: 793-797Crossref PubMed Scopus (286) Google Scholar). In experiments extending the time of Ba2+ addition by 15 min (not shown), the effect of TRPC7 knock out on the rate of Ba2+ entry was diminished, although still statistically significant (wild type, 0.063 + 0.008 ratio units/min, n = 197; clone 14.3, 0.040 + 0.006 ratio units/min, n = 205; p = 0.0213). In addition, in fluorescence experiments, membrane potential is not controlled, and a partially inhibited response could result from alterations in the driving force for entry. Thus, we next turned to analysis of the store-operated current Icrac (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar, 23Hoth M. Penner R. Nature. 1992; 355: 353-355Crossref PubMed Scopus (1486) Google Scholar) using whole-cell patch clamp. With this technique, Ca2+ stores can be rapidly depleted by a combination of IP3 and a Ca2+ chelator, BAPTA, in the patch pipette. As shown in Fig. 3, Icrac activation in TRPC7-/- cells did not differ significantly from that in wild-type cells. Note, however, that as reported by others, the magnitude of Icrac is rather small in these cells (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar, 15Prakriya M. Lewis R.S. J. Physiol. (Lond.). 2001; 536: 3-19Crossref Scopus (423) Google Scholar, 16Kiselyov K. Shin D.M. Shcheynikov N. Kurosaki M. Muallem S. Biochem. J. 2001; 360: 17-22Crossref PubMed Scopus (56) Google Scholar), such that a small, partial inhibitory effect of TRPC7 knock out would not likely be detected. However, this would contrast sharply with the almost complete loss of a non-store-operated pathway, documented below. Non-store-operated Entry in DT40 Wild-type and TRPC7-/- Cells— We next considered that TRPC7-/- cells might be deficient in non-store-operated Ca2+ entry, which is known to occur in this cell line (8Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (54) Google Scholar, 9Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.-G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In these experiments, we examined agonist activation of Ba2+ entry in the presence of 500 nm Gd3+, which is known to block completely the store-operated channels in DT40 cells (24Trebak 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). Fig. 4 shows responses to activation of the endogenous B-cell receptors with an anti-IgM. As reported previously, in response to B-cell receptor activation in the absence of extracellular Ca2+, DT40 cells exhibit irregular oscillations that dissipate gradually. The addition of 10 mm Ba2+, in the presence of 500 nm Gd3+, reveals the presence of a Ba2+-permeable pathway activated through the B-cell receptor (0.051 + 0.006 ratio units/min, n = 161). In TRPC7-/- cells, the oscillations were larger, more frequent, and more persistent presumably because of the increased capacity of intracellular Ca2+ stores (see Fig. 2). However, the addition of 10 mm Ba2+ did not result in a detectable elevation in basal fluorescence ratio in any of the TRPC7-/- cells (n = 183), indicating that the B-cell receptor-dependent Ba2+ entry was largely absent in these cells. The irregular pattern of Ca2+ oscillations following B-cell receptor activation makes quantitative analysis of the Ba2+ entry difficult. Thus, we also examined signaling through a G-protein-activated pathway, the protease-activated receptor 2 (PAR2), known to be expressed in this cell line (25Morita T. Tanimura A. Nezu A. Kurosaki T. Tojyo Y. Biochem. J. 2004; 382: 793-801Crossref PubMed Scopus (18) Google Scholar). As shown in Fig. 5, treatment of DT40 cells with 200 nm trypsin, which activates PAR2s, resulted in release of intracellular stores and Gd3+-insensitive entry of Ba2+. This Ba2+ entry was absent in TRPC7-/- cells (n = 136). Interestingly, the release of Ca2+ by trypsin did not appear to be augmented in the TRPC7-/- cells; this may indicate that this receptor type recruits a very specific endoplasmic reticulum Ca2+ pool. We examined the ability of the G-protein pathway to activate membrane currents likely responsible for the Gd3+-insensitive Ba2+ entry that appears to depend on TRPC7. Because not all cells respond to either B-cell receptor ligation (∼55–60%) or to PAR2 (∼55–70%), we activated G-proteins directly by including GTPγS in the patch pipette solution, while examining whole cell currents in the whole-cell mode. Following break-in with a pipette solution containing 300 μm GTPγS, a current developed that reversed near 0 mV and was slightly outwardly rectifying (Fig. 6, A and B; maximum currents were: outward, 14.7 + 1.2 picoamperes/picofarads; inward, -8.7 + 0.9 picoamperes/picofarads, n = 7). This behavior is reminiscent of the behavior of ectopically transfected TRPC7 (4Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). The current was largely absent in TRPC7-/- cells (Fig. 6A, n = 6). In its non-store-operated mode, TRPC7 can also be activated by synthetic diacylglycerols (4Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 6Lievremont J.P. Bird G.S. Putney Jr., J.W. Am. J. Physiol. 2004; 287: C1709-C1716Crossref PubMed Scopus (69) Google Scholar). However, the addition of the synthetic diacylglycerol OAG failed to activated any significant Ba2+ entry in wild-type or TRPC7-/- cells (Fig. 7, top). TRPC7 is known to be potently inhibited by protein kinase C (4Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), which would also be activated by OAG, perhaps to a greater extent than with agonist or GTPγS activation. Thus, we utilized a combination of two protein kinase C inhibitors, Gö6976 (1 μm) and calphostin C (0.5 μm), and under these conditions, OAG activated significant Ba2+ entry in wild-type DT40 cells (Fig. 7, bottom) and activated a current similar to that seen with GTPγS (Fig. 6C). The OAG-induced Ba2+ entry was significantly reduced in TRPC7-/- cells (Fig. 7, bottom) (wild type, 0.048 + 0.005 ratio units/min, n = 170; clone 14.3, 0.012 + 0.005, n = 183; p < 0.0001). We next sought to rescue the effects of TRPC7 deletion by transiently transfecting TRPC7-/- cells with a plasmid encoding for human TRPC7 (6Lievremont J.P. Bird G.S. Putney Jr., J.W. Am. J. Physiol. 2004; 287: C1709-C1716Crossref PubMed Scopus (69) Google Scholar). Initially, we used a rather high concentration of the pcDNA3 plasmid (100 μg/ml) because of the low expression levels generally obtained with the cytomegalovirus promoter (14Vazquez G. Wedel B.J. Trebak M. Bird G.St.J. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 21649-21654Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Transient expression of human TRPC7 resulted in a Ba2+ entry that could be activated by either a receptor agonist or OAG, but did not reverse the increased capacity of intracellular Ca2+ stores (not shown). The appearance of receptor- and OAG-activated entry following transient overexpression of TRPC7 is perhaps not surprising, given previous reports that overexpression of the close structural relative TRPC3 leads to similar behavior (14Vazquez G. Wedel B.J. Trebak M. Bird G.St.J. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 21649-21654Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 26Venkatachalam 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). The OAG-activated entry seen in TRPC7-/- cells transiently transfected with TRPC7 differed from OAG-activated entry in wild-type cells; in the TRPC7-transfected TRPC7-/- cells, OAG could activate entry even in the absence of protein kinase C (PKC) inhibitors (not shown). Thus, we next transfected TRPC7-/- cells with a lower concentration of TRPC7 encoding plasmid (10 μg/ml); in our previous work, this concentration of TRPC3 plasmid did not produce any additional receptoror OAG-activated entry (14Vazquez G. Wedel B.J. Trebak M. Bird G.St.J. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 21649-21654Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 24Trebak 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). With this apparently lower level of expression, neither wild-type nor TRPC7-/- cells responded to OAG in the absence of PKC inhibitors, whether transfected with TRPC7 or not (Fig. 8, A and C). In the presence of PKC inhibitors, the wild-type response was slightly but not significantly potentiated (Fig. 8B) (mock-transfected, 0.050 + 0.005 ratio units/min, n = 59; TRPC7-transfected, 0.053 + 0.004 ratio units/min, n = 65; p = 0.64); more significantly, in the TRPC7-/- cells, the response to OAG was restored by transfection with human TRPC7 (Fig. 8D) (mock-transfected, 0.017 + 0.006 ratio units/min, n = 51; TRPC7 transfected, 0.049 + 0.007 ratio units/min, n = 77; p = 0.016). Knock out of TRPC7 from DT40 B-lymphocytes produced three apparent phenotypes: an increased size of the Ca2+ stores, a diminished store-operated entry, and a diminished receptor- and diacylglycerol-activated entry. Of the three, only the latter, diacylglycerol-activated entry can be confidently attributed to the function of TRPC7 in native DT40 B-cells. The apparent decrease in store-operated entry appears to be secondary to the larger Ca2+ stores and the greater difficulty in completely depleting these stores with the sarco(endo)plasmic reticulum CA2+ ATPase inhibitor, thapsigargin. Thus, when stores were more rapidly depleted with a combination of IP3 and calcium chelator, the development of the store-operated current Icrac was not significantly different in wild-type and TRPC7-/- cells. It has previously been argued that TRPC channels can function in some environments as either store-operated or non-store-operated channels (1Putney Jr., J.W. Trends Cell Biol. 2004; 14: 282-286Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and when ectopically expressed in HEK293 cells, TRPC7 is clearly capable of functioning in both of these modes (6Lievremont J.P. Bird G.S. Putney Jr., J.W. Am. J. Physiol. 2004; 287: C1709-C1716Crossref PubMed Scopus (69) Google Scholar). Although the simplest interpretation of our data is that TRPC7 does not play an obligatory role in forming the Ca2+ release-activated Ca2+ channel in DT40 B-cells, it remains a possibility that, following knock out of the gene, other TRPC subunits may increase and assume this role. In addition, it is also conceivable that TRPC7 may function as a store-operated channel in other cell types, for example, in instances when the store-operated channels have conduction properties more akin to TRPCs (27Trepakova 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 (131) Google Scholar). The increased size of intracellular Ca2+ stores might indicate a role for TRPC7 in maintaining endoplasmic reticulum Ca2+ homeostasis, perhaps by functioning as either a constitutively active or regulated Ca2+-permeable channel. This would not be the first example of a TRPC capable of gating Ca2+ fluxes in endoplasmic reticulum as well as in the plasma membrane (28Turner H. Fleig A. Stokes A. Kinet J.P. Penner R. Biochem. J. 2003; 371: 341-350Crossref PubMed Scopus (99) Google Scholar, 29Wisnoskey B.J. Sinkins W.G. Schilling W.P. Biochem. J. 2003; 372: 517-528Crossref PubMed Scopus (61) Google Scholar). It is also not the first example of TRPC knock out affecting endoplasmic reticulum Ca2+ signaling. Mori et al. (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar) found that knock out of TRPC1 resulted in reduced sensitivity of endoplasmic reticulum stores to IP3-induced Ca2+ release. However, in that case, the thapsigargin-sensitive stores appeared unchanged. In the current study, the effects of TRPC7 knock out on calcium stores was not reversed by transient restoration of TRPC7. This may indicate that the effects on Ca2+ stores are more complex than simply loss of TRPC7 and may involve other longer term compensatory changes. On the other hand, the disappearance of a receptor- and diacylglycerol-activated Ba2+ entry and cation current was restored by transient transfection with a plasmid encoding for human TRPC7. Such an entry in DT40 cells has been reported in previous studies (8Vazquez G. Wedel B.J. Bird G.St.J. Joseph S.K. Putney Jr., J.W. EMBO J. 2002; 21: 4531-4538Crossref PubMed Scopus (54) Google Scholar, 9Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.-G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 30van Rossum D.B. Patterson R.L. Kiselyov K. Boehning D. Barrow R.K. Gill D.L. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2323-2327Crossref PubMed Scopus (59) Google Scholar) and appears to depend in some manner on IP3 receptors. However, the precise role of IP3 receptors in this pathway is controversial. In the present study, we demonstrated for the first time that (i) this entry appears to involve non-selective cation channels similar to TRPCs, (ii) this entry and current can be activated by exogenously applied diacylglycerols, and (iii) this entry and current are almost completely lost in the absence of TRPC7. The latter observation ties together the Ba2+ entry and currents seen under the various conditions of B-cell receptor activation, PAR2 activation, activation by intracellular application of GTPγS, and activation by OAG, as all of these responses were lost in the TRPC7-/- cells. This represents, to our knowledge, the first demonstration of a physiological function of endogenously expressed TRPC7. The broader role of non-store-operated TRPC7 channels in B-lymphocyte function cannot be determined at present. However, there is ample evidence for such channels in both T- and B-lymphocytes (31Grafton G. Thwaite L. Immunology. 2001; 104: 119-126Crossref PubMed Scopus (58) Google Scholar). The identity of a candidate channel subunit participating specifically in this pathway should open the way for more detailed analyses of the physiological role of receptor-regulated TRPC channels in B-lymphocytes and other non-excitable cells. The human TRPC7 plasmid was jointly supplied by Christine Murphy and Adrian Wolstenholm of the University of Bath and John Westwick of Novartis, Horsham, UK. Drs. David Miller and David Armstrong read the manuscript and provided helpful comments.
Referência(s)