20-Hydroxyeicosatetraenoic Acid (20-HETE) Activates Mouse TRPC6 Channels Expressed in HEK293 Cells

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 μm Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1–10 μm) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 μm) showed outward rectification, as shown previously for these channels using 100 μm 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 μm. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels ∼3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6. In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 μm Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1–10 μm) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 μm) showed outward rectification, as shown previously for these channels using 100 μm 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 μm. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels ∼3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6. Cationic channels thought to be responsible for capacitative Ca2+ entry, as originally defined by Putney (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2077) Google Scholar, 2Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1255) Google Scholar), have been identified in the plasmalemmal membrane of a spectrum of cells (3Fasolato C. Innocenti B. Pozzan T. Trends Pharmacol. Sci. 1994; 15: 77-83Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 4Hofmann T. Schaefer M. Schultz G. Gudermann T. J. Mol. Med. 2000; 78: 14-25Crossref PubMed Scopus (117) Google Scholar). Capacitative Ca2+ entry, through store-operated channels (SOCs) 1The abbreviations used are: SOC, store-operated channels; TRP, transient receptor potential; TRPC, transient receptor potential canonical; TRPL, TRP-like; 20-HETE, 20-hydroxyeicosatetraenoic acid; AA, arachidonic acid; NMDG, N-methyl-d-glucamine; DAG, diacylglycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; ETYA, 5,8,11,14-eicosatetraynoic acid; FFA, flufenamate; EET, eicosatrienoic acid; PLC, phospholipase C; m, mouse; d, Drosophila; h, human.1The abbreviations used are: SOC, store-operated channels; TRP, transient receptor potential; TRPC, transient receptor potential canonical; TRPL, TRP-like; 20-HETE, 20-hydroxyeicosatetraenoic acid; AA, arachidonic acid; NMDG, N-methyl-d-glucamine; DAG, diacylglycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; ETYA, 5,8,11,14-eicosatetraynoic acid; FFA, flufenamate; EET, eicosatrienoic acid; PLC, phospholipase C; m, mouse; d, Drosophila; h, human. is triggered by the emptying of intracellular Ca2+ stores by a variety of maneuvers (4Hofmann T. 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Overexpression studies have demonstrated that these channels show important differences in a number of properties including their mode of activation, unitary conductance, and selectivity, which may or may not be a result of the expression system used (for reviews, see Refs. 4Hofmann T. Schaefer M. Schultz G. Gudermann T. J. Mol. Med. 2000; 78: 14-25Crossref PubMed Scopus (117) Google Scholar, 17Birnbaumer 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 (352) Google Scholar, and 19Zitt C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. (N. Y.). 2002; 66: 243-264Crossref PubMed Scopus (122) Google Scholar). Moreover, the formation of heteromeric TRPC channels has been reported, whose characteristics were quite distinct from those recorded for the individual, homomeric channels; such behavior was shown for coassembly of TRPC1/TRPC3 (20Lintschinger B. 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Salvail D. Rousseau E. Am. J. Physiol. 2001; 280: L965-L973PubMed Google Scholar, 31Frisbee J.C. Roman R.J. Krishna U.M. Falck J.R. Lombard J.H. Am. J. Physiol. 2001; 280: H1066-H1074Crossref PubMed Google Scholar), the molecular mode of action of 20-HETE has yet to be established. It has been suggested that this compound represents an electropharmacological modulator capable of constricting various vascular and airway smooth muscle tissues by causing membrane depolarization and contraction (31Frisbee J.C. Roman R.J. Krishna U.M. Falck J.R. Lombard J.H. Am. J. Physiol. 2001; 280: H1066-H1074Crossref PubMed Google Scholar, 32Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) Google Scholar). Since several recent studies have determined that vascular smooth muscle cells express TRPC6 channels (33Walker R.L. Hume J.R. Horowitz B. Am. J. Physiol. 2001; 280: C1184-C1192Crossref PubMed Google Scholar, 34Jung S. Strotmann R. Schultz G. Plant T.D. Am. J. Physiol. 2002; 282: C347-C359Crossref PubMed Scopus (222) Google Scholar), the present study was aimed at assessing the electrophysiological effects of 20-HETE on mTRPC6 channels in an overexpression system. The rationale was to test whether 20-HETE might activate a specific isoform of TRPC conductances in an attempt to explain its well characterized inotropic effects, which are not directly related to the activation of 3,4-dihydro-2H-pyran-sensitive Ca2+ channels but rather to changes in non-selective membrane conductances (34Jung S. Strotmann R. Schultz G. Plant T.D. Am. J. Physiol. 2002; 282: C347-C359Crossref PubMed Scopus (222) Google Scholar, 35Welsh D.G. Morielli A.D. Nelson M.T. Brayden J.E. Circ. Res. 2002; 90: 248-250Crossref PubMed Scopus (414) Google Scholar). Our results conclusively demonstrate that application of 20-HETE activates mTRPC6 channels stably expressed in HEK293 cells and in smooth muscle cells. Cell Culture—HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 1 mm glutamine (Invitrogen). HEK-t6.11 stable clones were obtained as described previously (36Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) and were cultured in the same medium supplemented with 400 μg/ml G418 (Invitrogen). Smooth Muscle Cell Culture—Male or female albino were by a of and by The was and on The was of and on the of the smooth The cells were with a The smooth muscle was in medium μm Ca2+, and at for 1 The was and in μm Ca2+ with and μg/ml from The was at for The cell was through cell and the was with μm Ca2+ The cells were for and the was in 1 of supplemented by fetal bovine serum and The cells were in with cells for and at the were with of were used for in HEK293 and HEK-t6.11 cells were with the A. E. B. Arch. J. Physiol. PubMed Scopus Google Scholar) at using of were recorded with an by The potential was and membrane were at and at Single channel were in the inside-out with The membrane potential was to and the was at and at In whole-cell the intracellular mm 1 mm mm mm mm mm mm and mm Ca2+, 100 The mm 1.8 mm mm mm mm In inside-out a mm mm mm 1 mm mm was used in the and were by a system to their or μm 20-HETE, and 100 μm μm AA, μm 5,8,11,14-eicosatetraynoic acid 100 μm flufenamate and 1 μm the effects of 100 μm a was In some was with The of 100 μm was tested by cells for to were obtained from 20-HETE, AA, ETYA, and acid and was with a in is expressed as for are expressed as were on HEK-t6.11 cells stably as described previously (36Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). whole-cell an inward current was with a of = at a membrane potential of Application of 1 μm 20-HETE induced a in as shown in This current remained activated in the of was to a the of current rapidly to and were the current to relationship of the It is that the current an a slight inward and as the potential was found to be = a non-selective that the current was specific to mTRPC6 were also in on cells. The current in the of was not = from that recorded for HEK-t6.11 but the current was not increased by the application of 1 μm 20-HETE and the curves obtained in and 20-HETE were The relationship 20-HETE concentrations and current was using the cells. induced by 20-HETE, at were as the the current in the of and the current in the of the concentrations of The results in show that the effects of 20-HETE are with an EC50 of 0.8 μm. and that for a concentration of μm 20-HETE, not was the of the current at but the was Indeed, the was to has been described for TRPC6 T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1229) Google Scholar). the and in we using concentrations of OAG, a of 100 μm was the of the induced current at was = and the demonstrated a concentration = of the was to that obtained for 20-HETE concentrations or to showed inward The using the same and was found to be 100 μm = = μm = = μm 20-HETE = = results were obtained by which to through a the of and The activated current was and the was not to that recorded using concentrations of were triggered in HEK293 cells application of or not were in patches in the inside-out to whether 20-HETE could activate mTRPC6 in a membrane-delimited as shown for DAG T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1229) Google Scholar). Application of 1 μm 20-HETE to the induced a of the channel = of the is the number of and is the channel open increased results were obtained with 100 μm but with a in channel not the the ionic of the medium was were was by as the cation that the current was also in these The curves with or are in and that the potential was to = with in the The were in a low Ca2+ and as shown in C and of a 20-HETE 1.8 mm Ca2+ the of the current to that Ca2+ the current = In to Ca2+, several and can also TRPC Gd3+ has been used to capacitative at a concentration of 1 whereas 100 μm current activation by a 1 μm 20-HETE, the of 100 μm Gd3+ inhibited the current = whereas of 1 μm Gd3+ was not The cation channel was shown to by whereas by and were inhibited R. T. H. Y. Shimizu S. S. Y. Mori Y. Circ. Res. 2001; PubMed Scopus Google Scholar). to this we found that the current in HEK-t6.11 cells was not to 100 μm = In the effects of 20-HETE are inhibited by with an of that a metabolite of 20-HETE could be the compound M. K. R.M. E.R. Am. J. Physiol. 1997; 272: Google Scholar). tested whether this could be to system. that of HEK-t6.11 cells with 1 μm for did not the current = In smooth muscle has been reported that activated a Ca2+ directly by arachidonic acid produced by the of and DAG lipase L.M. Cannon T.R. Taylor C.W. J. Physiol. 1999; 517: 121-134Crossref PubMed Scopus (194) Google Scholar). channels are activated by DAG and DAG and by including and C. Halaszovich C.R. Luckhoff A. Prog. Neurobiol. (N. Y.). 2002; 66: 243-264Crossref PubMed Scopus (122) Google Scholar, S. Hardie R.C. Nature. 1999; 397: PubMed Scopus Google Scholar). In μm) activated a current with properties to those of the current A and = Application of a ETYA, at a concentration of μm did not C and = The concentration of used of including phospholipase A2, and cytochrome that the are to of its Our was to whether 20-HETE could a in a cell that TRPC6 tested of smooth muscle cells obtained from expression of TRPC6 was by not In we show that 1 μm 20-HETE induced an inward current in these cells with an of = Interestingly, of 20-HETE did not result in of the induced The current-voltage relationship is to that obtained with 20-HETE in cells. The and conclusively show that the eicosanoid compound, 20-HETE, activates mTRPC6 channels in cells. TRPC6 channels to the (4Hofmann T. Schaefer M. Schultz G. Gudermann T. J. Mol. Med. 2000; 78: 14-25Crossref PubMed Scopus (117) Google Scholar), which the of activated by DAG or Application of μm) activated a current a in cells T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. 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Am. J. Physiol. 2002; 282: C347-C359Crossref PubMed Scopus (222) Google Scholar, R. T. H. Y. Shimizu S. S. Y. Mori Y. Circ. Res. 2001; PubMed Scopus Google Scholar). we did not that on the have shown that TRPC members can with from the same The heteromeric channels were shown to homomeric channels with that the overexpression of mTRPC6 did not result in the formation of such heteromeric channels by studies not Moreover, the expression of the various TRPC were not and cells not have shown that in AA, could in smooth muscle cells and could activate the related In of induced non-selective to those induced by Moreover, of current activation using a of the that action to be by a metabolite, Indeed, is by cytochrome P-450 enzymes to and and 20-hydroxyeicosatetraenoic and It is now that these a as in the of vascular (27Roman R.J. Alonso-Galicia M. News Physiol. Sci. 1999; 14: 238-242PubMed Google Scholar). 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