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• W1976284742 abstract "Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPγ, has been identified and described as an auxiliary subunit of TRPL. Beyond it, our data show that heterologously expressed TRPγ formed a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPγ is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A2-dependent manner. The most potent activator of TRPγ, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Here we show that upon heterologous expression TRPγ forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPγ is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPγ forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades. Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPγ, has been identified and described as an auxiliary subunit of TRPL. Beyond it, our data show that heterologously expressed TRPγ formed a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPγ is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A2-dependent manner. The most potent activator of TRPγ, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Here we show that upon heterologous expression TRPγ forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPγ is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPγ forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades. The number of G-protein-coupled receptors functionally expressed in Drosophila is still elusive. However, the access to the sequence of the entire genome for Drosophila melanogaster allowed the categorization of Drosophila G-protein-coupled receptors (GPCR) 2The abbreviations used are: GPCR, G-protein-coupled ; HEK, human embryonic kidney; PUFA, polyunsaturated fatty acid; AA, arachidonic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; MDG, 1-decanoyl-rac-glycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; DOG, 1,2-dioctanoyl-sn-glycerol; RT-PCR, reverse transcription PCR; YFP, yellow fluorescent protein; NMDG, N-methyl-d-glucamine; PLA2, phospholipase A2. 2The abbreviations used are: GPCR, G-protein-coupled ; HEK, human embryonic kidney; PUFA, polyunsaturated fatty acid; AA, arachidonic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; MDG, 1-decanoyl-rac-glycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; DOG, 1,2-dioctanoyl-sn-glycerol; RT-PCR, reverse transcription PCR; YFP, yellow fluorescent protein; NMDG, N-methyl-d-glucamine; PLA2, phospholipase A2. (1Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.H. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C. Baxter E.G. Helt G. Nelson C.R. Gabor G.L. Abril J.F. Agbayani A. An H.J. Andrews-Pfannkoch C. Baldwin D. Ballew R.M. Basu A. Baxendale J. Bayraktaroglu L. Beasley E.M. Beeson K.Y. Benos P.V. Berman B.P. Bhandari D. Bolshakov S. Borkova D. Botchan M.R. Bouck J. Brokstein P. Brottier P. Burtis K.C. Busam D.A. Butler H. Cadieu E. Center A. Chandra I. Cherry J.M. Cawley S. Dahlke C. Davenport L.B. Davies P. de Pablos B. Delcher A. Deng Z. Mays A.D. Dew I. Dietz S.M. Dodson K. Doup L.E. Downes M. Dugan-Rocha S. Dunkov B.C. Dunn P. Durbin K.J. Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H. Gu Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houston K.A. Howland T.J. Wei M.H. Ibegwam C. Jalali M. Kalush F. Karpen G.H. Ke Z. Kennison J.A. Ketchum K.A. Kimmel B.E. Kodira C.D. Kraft C. Kravitz S. Kulp D. Lai Z. Lasko P. Lei Y. Levitsky A.A. Li J. Li Z. Liang Y. Lin X. Liu X. Mattei B. McIntosh T.C. McLeod M.P. McPherson D. Merkulov G. Milshina N.V. Mobarry C. Morris J. Moshrefi A. Mount S.M. Moy M. Murphy B. Murphy L. Muzny D.M. Nelson D.L. Nelson D.R. Nelson K.A. Nixon K. Nusskern D.R. Pacleb J.M. Palazzolo M. Pittman G.S. Pan S. Pollard J. Puri V. Reese M.G. Reinert K. Remington K. Saunders R.D. Scheeler F. Shen H. Shue B.C. Siden-Kiamos I. Simpson M. Skupski M.P. Smith T. Spier E. Spradling A.C. Stapleton M. Strong R. Sun E. Svirskas R. Tector C. Turner R. Venter E. Wang A.H. Wang X. Wang Z.Y. Wassarman D.A. Weinstock G.M. Weissenbach J. Williams S.M. Woodage T. Worley K.C. Wu D. Yang S. Yao Q.A. Ye J. Yeh R.F. Zaveri J.S. Zhan M. Zhang G. Zhao Q. Zheng L. Zheng X.H. Zhong F.N. Zhong W. Zhou X. Zhu S. Zhu X. Smith H.O. Gibbs R.A. Myers E.W. Rubin G.M. Venter J.C. Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4741) Google Scholar). More than 100 genes coding for putative GPCRs were identified, including 22 genes encoding receptors for biogenic amines and 32 genes encoding receptors for peptides (2Brody T. Cravchik A. J. Cell Biol. 2000; 150: F83-F88Crossref PubMed Scopus (213) Google Scholar, 3Hewes R.S. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (448) Google Scholar). Many of the Drosophila receptors have been characterized in human embryonic kidney (HEK) 293 cells showing that signaling cascades found in mammalian cells generating second messengers like cAMP or increases in intracellular Ca2+ concentrations are also induced by Drosophila GPCRs. The application of ligands like octopamine or leucokinine to HEK293 cells expressing the Dmoa1 or CG10626 receptors ubiquitously expressed in Drosophila resulted in increased intracellular Ca2+ (4Balfanz S. Strunker T. Frings S. Baumann A. J. Neurochem. 2005; 93: 440-451Crossref PubMed Scopus (135) Google Scholar, 5Radford J.C. Davies S.A. Dow J.A. J. Biol. Chem. 2002; 277: 38810-38817Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, it is likely that not only sensory processes but also many other physiological functions in Drosophila, e.g. development (4Balfanz S. Strunker T. Frings S. Baumann A. J. Neurochem. 2005; 93: 440-451Crossref PubMed Scopus (135) Google Scholar) and hindgut motility and renal fluid secretion (5Radford J.C. Davies S.A. Dow J.A. J. Biol. Chem. 2002; 277: 38810-38817Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), depend on changes in intracellular Ca2+ concentration mediated by release from intracellular calcium stores or by influx mechanism. Drosophila TRP was the first protein described mediating Ca2+ influx in Drosophila photoreceptor cells in response to activation of a GPCR (6Montell C. Rubin G.M. Neuron. 1989; 2: 1313-1323Abstract Full Text PDF PubMed Scopus (847) Google Scholar). Since the identification of Drosophila TRP a large number of homologous proteins have been cloned. Today, TRP proteins form a superfamily of nonselective cation channels containing six putative transmembrane domains, a pore region between the fifth and sixth segment, and cytosolic C and N termini (7Clapham D.E. Julius D. Montell C. Schultz G. Pharmacol. Rev. 2005; 57: 427-450Crossref PubMed Scopus (319) Google Scholar). Based on sequence analysis of genomic and expressed sequence tag data, three different groups of TRP channels, TRPC (C for “classic” or “canonical”), TRPV (V for “vanilloid receptorlike”), and TRPM for (M for “melastatin-like”), have been identified (8Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 9Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). In addition, other related channel protein families have been classified as TRP channels by phylogenetic analysis (TRPN, TRPA, TRPML, TRPP) (10Montell C. Science's STKE. 2005; 2005: re3PubMed Google Scholar, 11Yu F.H. Catterall W.A. Science's STKE. 2004; 2004: re15Crossref PubMed Scopus (337) Google Scholar). Thirteen TRP channels, including the TRPC members TRP and TRPL, have been identified in the Drosophila genome so far with their biological functions mainly related to sensory systems (12Montell C. Pflügers Arch. 2005; 451: 19-28Crossref PubMed Scopus (95) Google Scholar). Mammalian and Drosophila TRPC channels are activated by mediators created by the GPCR-dependent stimulation of phospholipase C isoforms. Whereas the mammalian TRPC channels TRPC3, TRPC6, and TRPC7 (13Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1229) Google Scholar, 14Okada 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 (396) Google Scholar) and TRPC2 (15Lucas P. Ukhanov K. Leinders-Zufall T. Zufall F. Neuron. 2003; 40: 551-561Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) are activated by diacylglycerols, Drosophila TRPL, and possibly TRP, are activated by the polyunsaturated fatty acids (PUFA), arachidonic acid (AA) and linoleic acid (16Chyb S. Raghu P. Hardie R.C. Nature. 1999; 397: 255-259Crossref PubMed Scopus (356) Google Scholar). A third Drosophila TRPC channel, TRPγ, was identified and characterized as an auxiliary subunit of TRPL that, when co-expressed with TRPL in HEK293 cells, forms a receptor-activated cation channel (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). However, the activation mechanism of TRPγ remained obscure. TRP and TRPL and possibly TRPγ participate in phototransduction. The distribution of TRPγ expression in Drosophila, however, is controversial. Xu et al. showed expression of TRPγ predominantly in Drosophila head (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In contrast, two recent publications showed a much broader distribution of TRPγ (18MacPherson M.R. Pollock V.P. Kean L. Southall T.D. Giannakou M.E. Broderick K.E. Dow J.A. Hardie R.C. Davies S.A. Genetics. 2005; 169: 1541-1552Crossref PubMed Scopus (25) Google Scholar, 19Wicher D. Agricola H.J. Schönherr R. Heinemann S.H. Derst C. J. Biol. Chem. 2006; 281: 3227-3236Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A broad expression pattern of TRPγ and the fact that there are only three TRPC members (TRP, TRPL, TRPγ) in the genome of Drosophila make it likely that a TRPγ-mediated Ca2+ influx is integrated in many receptor-mediated signaling pathways outside the visual system. In our study, we show that TRPγ is expressed in heads and in the bodies of fruitflies. When heterologously expressed in HEK293 cells, TRPγ forms a channel that is regulated via a hormone-induced, GPCR-activated, intracellular signaling pathway. Analysis of this signaling pathway revealed that TRPγ is activated by polyunsaturated fatty acids in a phospholipase C- and phospholipase A2-dependent manner. The activation of TRPγ by 5,8,11,14-eicosatetraynoic acid (ETYA) excludes the participation of metabolites of arachidonic acid. In summary, we show, for the first time, the functional and biophysical characterization of TRPγ as a homomeric, non-selective cation channel that is activated by polyunsaturated fatty acids. Chemicals—AA, linoleic acid (Sigma), and palmitoleic acid (MP Biochemical, Heidelberg, Germany) were diluted from 100-mm stock solutions in ethanol. 1-decanoyl-rac-glycerol (MDG), 1-oleoyl-rac-glycerol, 1,2-dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2-acetyl-sn-glycerol (OAG) (Sigma) were used from 100-mm stock solution in dimethyl sulfoxide (Me2SO). The phospholipase A2 inhibitors N-(p-amylcinnamoyl) anthranilic acid, arachidonyltrifluoromethyl ketone (Calbiochem), bromoenol lactone (Sigma) were diluted from 50-mm stock solutions in Me2SO. p-bromphenacyl bromide (pBPB) (Sigma), ETYA (Calbiochem) were used from 50-mm stock solution in ethanol. Extraction of mRNA and RT-PCR—Total RNA was isolated from wild-type D. melanogaster using TriReagent (Ambion, Austin, TX) according to the standard protocol and subsequent incubation with Turbo RNase-free DNase I (Ambion). The heads were separated from the body on dry ice before RNA preparation. cDNAs were generated using M-MLV reverse transcriptase Rnase H Minus (Promega) and served as templates in a subsequent PCR, using TaqPCR Master Mix (Qiagen, Hilden, Germany) and specific oligonucleotides (TRPγ: sense, 5′-AGTCGGAAACGTGAGCAAAATG-3′, and antisense, 5′-TGGAGTTCACTGACGTATTGGATG-3′; glyceraldehyde-3-phosphate dehydrogenase: sense, 5′-GTGCCCACGC CCAATGTCTCC-3′, and antisense, 5′-GGCGCCGGGTTTGTACGATAGTTT-3′). Molecular Cloning of TRPγ—The cDNAs coding for TRP and TRPL have been described earlier (20Zitt C. Zobel A. Obukhov A.G. Harteneck C. Kalkbrenner F. Lückhoff A. Schultz G. Neuron. 1996; 16: 1189-1196Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 21Harteneck C. Obukhov A.G. Zobel A. Kalkbrenner F. Schultz G. FEBS Lett. 1995; 358: 297-300Crossref PubMed Scopus (76) Google Scholar). The TRPγ cDNA was a kind gift from C. Montell (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The different plasmids were used as templates for amplification of the entire reading frames, omitting the endogenous stop codon by using the oligonucleotides (TRP sense, 5′-CACC ATGGGCAGCAATACGG-3′, and antisense, 5′-GAGCCAGCCGGAGATCAT-3′; TRPL sense, 5′-CACCATGGGACGCAAAAAGAAGCTGCCGACG-3′, and antisense, 5′-GTTTCTATGCTTTGGCCGCTGGGGACTCG-3′; TRPγ sense, 5′-CACCATGATGGAGGAGGAGAACAC-3′, and antisense, 5′-ACCGATAGCTCCCGTGGTAGAAACA-3′). The fragments were subcloned in the expression vector pcDNA3.1 Directional/V5-His-TOPO (Invitrogen), resulting in constructs of fusion proteins with C-terminal V5 and His6 tags. For expression as yellow fluorescence protein fusion protein, the coding sequence of YFP was subcloned in-frame C-terminal of the TRPC channel proteins. Both strands of all cDNA fragments were sequenced using ABI Prism BigDye terminator cycle sequencing kits and an ABI Prism 377 DNA sequencer (Applied Biosystems, Weiterstadt, Germany). DNA for transient transfection was prepared using anion exchange columns (Qiagen). Cell Culture and Transfection of HEK293 Cells—HEK293 cells were cultured in Earle's minimal essential medium (Biochrom, Berlin, Germany), supplemented with 10% fetal calf serum (Biochrom), 100 μg/ml penicillin, and 100 μg/ml streptomycin under a 5% CO2 humidified atmosphere at 37 °C. Cells were plated in 85-mm dishes onto glass coverslips and transiently transfected 2 days later by addition of a transfection mixture containing 2.5–3 μg of DNA and 7 μl of FuGENE 6 transfection reagent (Roche Diagnostics) in 93 μl of Opti-MEM medium (Invitrogen). Fluorescence measurements and electrophysiological studies were carried out 1–2 days after transfection. Western Blot Analysis—Transfected HEK293 cells were harvested by centrifugation (800 × g, 5 min, room temperature). Cells were resuspended in lysis buffer (50 mm Tris/HCl, 2 mm dithiothreitol, 0.2 μm benzamidine, 1 mm EDTA, pH 8.0) and homogenized by shearing through 26-gauge needles. After removal of nuclei (800 × g, 2 min, 4 °C), supernatants were mixed with gel loading buffer (62.5 mm Tris/HCl, 10% glycerol, 5% mercaptoethanol, 2% SDS, 0.02% bromphenol blue, pH 6.8). To detect TRPγ, TRP, and TRPL expressed in HEK293 cells, the membrane extracts were separated on an 8% SDS-PAGE (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). After electrophoresis the proteins were transferred on nitrocellulose membrane, and the fusion proteins were detected by incubating the membrane with an anti-tetra His monoclonal antibody 1:10000 at 4 °C overnight. The bound antibody was detected using an ECL Advance Western blotting detection kit (Amersham Biosciences). Fluorescence Measurements—[Ca2+]i measurements in single cells were carried out using the fluorescence indicator Fura-2/AM in combination with a monochromator-based imaging system (T.I.L.L. Photonics, Martinsried, Germany) attached to an inverted microscope (Axiovert 100; Carl Zeiss, Oberkochen, Germany). HEK293 cells were loaded with 4 μm Fura-2/AM (Molecular Probes) and 0.01% Pluronic F-127 (Molecular Probes) for 60 min at room temperature in a standard solution composed of 138 mm NaCl, 6 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 5.5 mm glucose, and 10 mm HEPES (adjusted to pH 7.4 with NaOH). The osmolarity of the solution amounted to 300 mosmol–1 and was measured using a freezing point depression osmometer (Roebling, Berlin, Germany). Coverslips were then washed in this buffer for 20 min and mounted in a perfusion chamber on the microscope stage. For [Ca2+]i measurements, fluorescence was excited at 340 and 380 nm. After correction for background fluorescence, the fluorescence ratio F340/F380 was calculated. Fluorescence quenching by Mn2+ entry was studied using the Fura-2 isosbestic excitation wavelength at 360 nm, and the emitted light was monitored using the same filter system as for [Ca2+]i measurements. In all experiments, transfected cells of the whole field of vision were identified by their YFP fluorescence at an excitation wavelength of 480 nm. Experiments with at least 20 cells were summarized and are given as the number of experiments for each experimental condition. Patch Clamp Measurements—Membrane currents were recorded using the whole-cell, cell-attached or inside-out configurations of the patch clamp technique at room temperature. Pipettes were made from borosilicate glass capillary tubes. The resistance of the pipettes varied between 2 and 5 mΩ in whole-cell recordings and between 7 and 9 mΩ in single channel recordings. Whole-cell currents were elicited by voltage ramps from –100 to +100 mV (400-ms duration) applied every 10 s from a holding potential of 0 mV. Currents through the pipette were recorded by an Axopatch 200B amplifier (Axon Instruments), filtered at 5 or 10 kHz (Bessel filter), and analyzed using pCLAMP software (version 9.2; Axon Instruments). Pipettes for whole-cell recordings were filled with a solution composed of 130 mm CsCH3O3S, 10 mm CsCl, 2 mm MgCl2, and 10 mm HEPES (pH 7.2 with CsOH). The standard bath solution contained 140 mm NaCl, 2 mm CaCl2, 1 mm MgCl2, 10mm glucose, and 10 mm HEPES (pH 7.4 with NaOH). For Na+- and divalent cation-free conditions, the bath solutions contained 140 mm N-methyl-d-glucamine (NMDG+) and 10 mm HEPES (pH 7.4 with HCl). Single channel currents were continuously measured at different pipette potentials. The corresponding membrane potentials for inside-out patches were calculated by the equation Vm =–Vp + VL, where Vm is the membrane potential and VL is the junction potential. Pipettes were either filled with 130 mm CsCH3O3S, 10 mm CsCl, 2 mm MgCl2, and 10 mm HEPES (pH 7.4 with CsOH) for inside-out recordings or with 130 mm NaCl, 2 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 7.4 with NaOH) for cell-attached recordings. Values for VL were calculated using pCLAMP software (version 9.2). Single channel amplitudes at different Vm values were calculated from current traces of 2–4 s using amplitude histograms fitted to Gaussian functions. Detection of TRPγ mRNA in Drosophila Heads and Residual Bodies—To clarify whether or not TRPγ expression is restricted to the head of Drosophila, we performed RT-PCR analysis from RNA isolated from Drosophila heads and adult residual bodies (Fig. 1A). The synthetic oligonucleotides for PCR were designed to distinguish fragments of different lengths depending on the template being amplified. A 656-bp fragment indicated an amplification starting from genomic DNA, whereas the 545-bp fragment corresponded to the TRPγ cDNA as template. The RT-PCR repeatedly resulted in the amplification of the 545-bp fragment from head and body cDNA (see Fig. 1A). Parallel RT-PCR reactions amplifying a fragment of glyceraldehyde-3-phosphate dehydrogenase were performed as control to ensure that comparable RNA amounts were used for the reactions (see Fig. 1A). Heterologous Expression of TRP, TRPL, and TRPγ in HEK293 Cells—To study the function of the Drosophila TRPC channels, we subcloned the cDNA fragments coding for TRP, TRPL, and TRPγ in a vector directing the expression of the channel proteins as C-terminal fusion proteins with either V5 tag and His tag, or YFP. We verified the capability of the constructs to direct expression of the TRPC proteins in HEK293 cells in Western blot analyses using an antibody directed against the C-terminal His tag (Fig. 1B). The apparent molecular masses of the expressed proteins were comparable with published data (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In membrane extracts of TRP-expressing HEK293 cells, the antibody detected two proteins with differences in apparent molecular mass. To test whether these differences resulted from posttranslational protein modifications, we incubated aliquots of the extracted membrane protein with Endo H glycosidase, PNGase F glycosidase, and λ protein phosphatase. Subsequent Western blot analysis demonstrated that the appearance of two TRP protein bands did not result from differences in glycosylation (supplemental Fig. S1) or from differences in protein phosphorylation. TRPγ Forms a Receptor-regulated Cation Channel—Next, we studied TRPγ in transiently transfected HEK293 cells. Fluorescence energy transfer (FRET) experiments using TRPγ-CFP and -YFP fusion proteins expressed in HEK293 cells resulted in FRET signals of ∼10% (supplemental Fig. S2), arguing for the formation of a homomeric channel complex. For functional characterization of this homomeric TRPγ channel complex, we initially applied Ca2+-imaging methods. In Fura-2-loaded cells, the expression of TRPγ resulted in an increase in the basal Ca2+ signal (Fig. 2A), due to its spontaneous activity (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). To test whether TRPγ is regulated by receptor activation, we applied carbachol to stimulate endogenous muscarinic receptors in HEK293 cells. To our surprise the application of carbachol increased the intracellular Ca2+ ([Ca2+]i) in TRPγ-expressing cells ∼10-fold as compared with control cells (see Fig. 2A). The increase in [Ca2+]i resulted from TRPγ-mediated Ca2+ entry, because it was suppressed by extracellular EGTA. The origin of the increased [Ca2+]i was further analyzed by modified protocols (Fig. 2B). In the absence of extracellular Ca2+ and in the presence of extracellular EGTA, carbachol induced only a small increase in [Ca2+]i. The addition of extracellular Ca2+ induced a pronounced Ca2+ influx into TRPγ-expressing cells. Manganese quenching of intracellular Fura-2 allows a direct correlation of a fluorescence signal and the activity of a plasma membrane channel protein. Application of manganese to TRPγ-expressing cells resulted in an instantaneous slow and progressive reduction of Fura-2 fluorescence due to the spontaneous activity of TRPγ (17Xu X.Z. Chien F. Butler A. Salkoff L. Montell C. Neuron. 2000; 26: 647-657Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) (Fig. 2C). Subsequent activation of muscarinic receptors after application of carbachol further enhanced the manganese quenching of Fura-2 fluorescence (see Fig. 2C). Based on the result that TRPγ is receptor regulated, we tested common blockers of TRP channels for further functional characterization. The application of Gd3+ (100 μm), La3+ (100 μm), or SKF-96365 (100 μm) completely blocked TRPγ-mediated manganese quench in TRPγ-expressing HEK293 cells (Fig. 2D). In good agreement with the increased basal intracellular Ca2+ concentration, we observed spontaneous currents in whole-cell recordings from TRPγ-expressing cells. Application of carbachol (100 μm) increased current responses in TRPγ-expressing cells. Currents returned to a level similar to that before the application of carbachol during wash-out phase and removal of the agonist (data not shown). The current-voltage relationship obtained from whole-cell currents before and after the application of carbachol displayed outward rectification (Fig. 3A) and a reversal potential of –4 ± 4mV(n = 11). The mean current densities under carbachol were –26 ± 16 pA/pF and +47 ± 7 pA/pF (n = 11) at –90 and +90 mV, respectively (Fig. 3B). Identical current-voltage relationships were measured under a voltage step protocol (10 mV/step from –70 to 70 mV) before (Fig. 3C) and after (Fig. 3D) application of carbachol. The plateau of maximal current was reached ∼40 s after carbachol application (Fig. 3E). The exchange of Na+ for the large cation NMDG+ in the bath solution resulted in a clear decrease in inward currents in TRPγ-transfected HEK293 cells (Fig. 3F). The NMDG+-sensitive inward current was –6 ± 7 pA pF–1 at –90 mV (n = 5) using a Ca2+-free, Mg2+-containing (2 or 5 mm) pipette solution. The current-voltage relationship of the current of spontaneously active TRPγ showed no clear rectification and a reversal potential (Erev) of –60 ± 20 mV (n = 10). To study the selectivity of the current of TRPγ in transfected cells, we replaced the standard extracellular solution containing Na+, Ca2+, and Mg2+ by solutions containing only one of the cations, NMDG+, Na+, Ca2+, or Mg2+. In Na+-containing bath solution the removal of divalent cations induced a slight increase in inward and outward currents (data not shown). Permeability ratios of TRPγ were PNa/PNMDG = 1:0.15, and PCs/PNa = 1:1 (n = 6). Analysis of the Signaling Pathway Leading to TRPγ Activation—To clarify whether TRPγ is activated by phospholipase C-generated mediators, we used the phospholipase C inhibitor U73122. In the presence of U73122, the application of carbachol to TRPγ-transfected HEK293 cells did not result in an increase in [Ca2+]i, suggesting a phospholipase C-dependent activation of TRPγ (data not shown). As some members of mammalian TRPC channels are activated by diacylglycerol, we used cell-permeable diacylglycerol analogues in our Ca2+ imaging experiments. We applied 1,2-dioctanoyl-sn-glycerol, OAG, as well as monoacylglycerols such as MDG, and 1-oleoyl-rac-glycerol to test their ability to induce a TRPγ-mediated Ca2+ entry in HEK293 cells transfected with TRPγ DNA (Fig. 4). Carbachol reproducibly induced an increase in intracellular Ca2+ in TRPγ-transfected cells, whereas OAG did not result in increases in [Ca2+]i either in TRPγ-transfected cells or in control cells (Fig. 4A). Due to the instability of OAG, we performed side-by-side experiments using TRPC6-expressing HEK293 cells as control to verify the integrity of OAG (Fig. 4B). Whereas the diacylglycerols selectively activated TRPC6, the monoacylglycerol analogues failed to stimulate TRPC6 as well as TRPγ (Fig. 4C). To determine the putative phospholipase C-dependent and diacylglycerol-independent activation of TRPγ, we looked for additional inhibitors interfering with phospholipase C-triggered pathways. In several reports, receptor-induced increases in arachidonic acid have been described and linked to the receptor-mediated activation of phospholipase A2 isoenzymes (23Leurs R. Traiffort E. Arrang J.M. Tardivel-Lacombe J. Ruat M. Schwartz J.C. J. Neurochem. 1994; 62: 519-527Crossref PubMed Scopus (100) Google Scholar, 24Husain S. Abdel-Latif A.A. Biochem. J. 1999; 342: 87-96Crossref PubMed Scopus (37) Google Scholar, 25Handlogten M.E. Huang C. Shiraishi N. Awata H. Miller R.T. J. Biol. Chem. 2001; 276: 13941-13948Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Therefore, we tested four PLA2 inhibitors (N(p-amylcinnamoyl)anthranilic acid, p-bromphenacyl bromide bromoenol lactone, and arachidonyltrifluoromethyl ketone) (Fig. 5). In manganese quench experiments," @default.
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• W1976284742 title "Receptor-induced Activation of Drosophila TRPγ by Polyunsaturated Fatty Acids" @default.
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