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Originally published In Press as doi:10.1074/jbc.M602215200 on August 10, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29693-29702, October 6, 2006
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Receptor-induced Activation of Drosophila TRP{gamma} by Polyunsaturated Fatty Acids*Formula

Simone Jörs{ddagger}, Victor Kazanski{ddagger}, Anna Foik{ddagger}§, Dietmar Krautwurst, and Christian Harteneck{ddagger}1

From the {ddagger}Institut für Pharmakologie, Charité Campus Benjamin Franklin, Thielallee 69-73, 14195 Berlin, §Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, and Abteilung für Molekulare Genetik, Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany

Received for publication, March 9, 2006 , and in revised form, July 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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, TRP{gamma}, has been identified and described as an auxiliary subunit of TRPL. Beyond it, our data show that heterologously expressed TRP{gamma} formed a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRP{gamma} is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A2-dependent manner. The most potent activator of TRP{gamma}, 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 TRP{gamma} 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 TRP{gamma} is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRP{gamma} forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)2 (1). 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 (2, 3). 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+ (4, 5). Therefore, it is likely that not only sensory processes but also many other physiological functions in Drosophila, e.g. development (4) and hindgut motility and renal fluid secretion (5), 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 (6). 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 (7). 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 (8, 9). In addition, other related channel protein families have been classified as TRP channels by phylogenetic analysis (TRPN, TRPA, TRPML, TRPP) (10, 11).

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 (12). 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 (13, 14) and TRPC2 (15) are activated by diacylglycerols, Drosophila TRPL, and possibly TRP, are activated by the polyunsaturated fatty acids (PUFA), arachidonic acid (AA) and linoleic acid (16). A third Drosophila TRPC channel, TRP{gamma}, 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 (17). However, the activation mechanism of TRP{gamma} remained obscure. TRP and TRPL and possibly TRP{gamma} participate in phototransduction. The distribution of TRP{gamma} expression in Drosophila, however, is controversial. Xu et al. showed expression of TRP{gamma} predominantly in Drosophila head (17). In contrast, two recent publications showed a much broader distribution of TRP{gamma} (18, 19). A broad expression pattern of TRP{gamma} and the fact that there are only three TRPC members (TRP, TRPL, TRP{gamma}) in the genome of Drosophila make it likely that a TRP{gamma}-mediated Ca2+ influx is integrated in many receptor-mediated signaling pathways outside the visual system.

In our study, we show that TRP{gamma} is expressed in heads and in the bodies of fruitflies. When heterologously expressed in HEK293 cells, TRP{gamma} forms a channel that is regulated via a hormone-induced, GPCR-activated, intracellular signaling pathway. Analysis of this signaling pathway revealed that TRP{gamma} is activated by polyunsaturated fatty acids in a phospholipase C- and phospholipase A2-dependent manner. The activation of TRP{gamma} 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 TRP{gamma} as a homomeric, non-selective cation channel that is activated by polyunsaturated fatty acids.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (TRP{gamma}: 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 TRP{gamma}—The cDNAs coding for TRP and TRPL have been described earlier (20, 21). The TRP{gamma} cDNA was a kind gift from C. Montell (17). 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'; TRP{gamma} 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 x 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 x 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 TRP{gamma}, TRP, and TRPL expressed in HEK293 cells, the membrane extracts were separated on an 8% SDS-PAGE (22). 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 M{Omega} in whole-cell recordings and between 7 and 9 M{Omega} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of TRP{gamma} mRNA in Drosophila Heads and Residual Bodies—To clarify whether or not TRP{gamma} 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 TRP{gamma} 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).


Figure 1
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  		FIGURE 1.
Expression of TRP{gamma}, TRP, and TRPL. A, cDNA samples from Drosophila heads and bodies were used as templates to amplify DNA fragments specific for TRP{gamma}. Amplification of a 545-bp fragment indicates that TRP{gamma} is expressed in Drosophila heads and bodies. The comparable intensities of the amplified bands coding of glyceraldehyde-3-phosphate dehydrogenase (322 bp) show that equal amounts of RNA from Drosophila heads and bodies have been used in the RT-PCR reactions. B, for Western blot analysis of heterologously expressed Drosophila TRPs, membrane proteins from non-transfected control HEK293 cells and TRP{gamma}-His-, TRP-His-, and TRPL-His-expressing cells were separated by SDS/PAGE electrophoresis and transferred on nitrocellulose membranes as described under "Experimental Procedures." Immobilized proteins were visualized by incubating with an anti-tetra-histidine antibody, followed by incubation with a secondary antibody coupled to peroxidase, and chemiluminescence detection.

 
Heterologous Expression of TRP, TRPL, and TRP{gamma} in HEK293 Cells—To study the function of the Drosophila TRPC channels, we subcloned the cDNA fragments coding for TRP, TRPL, and TRP{gamma} 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 (17). 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 {lambda} 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.


Figure 2
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  		FIGURE 2.
Activation of signaling-induced Ca2+ influx in TRP{gamma}-HEK293 cells. A, shown are effects of 100 µM carbachol (CCh) on [Ca2+]i in TRP{gamma}-YFP-expressing cells (n = 4) and YFP-expressing control cells (n = 3). During application of CCh, 2 mM Ca2+ was exchanged for 2 mM EGTA. B, carbachol (100 µM) was applied in the presence of 2 mM EGTA and after addition of 2 mM extracellular Ca2+. Shown are effects in TRP{gamma}-YFP-expressing cells (n = 4) and YFP-expressing cells (n = 3). C, influx of Mn2+ (1 mM extracellular) into TRP{gamma}-YFP-expressing cells (n = 8) was enhanced by addition of 100 µM CCh. YFP-expressing cells (n = 3) served as control. Panels A–C represent the averaged traces of one independent experiment (out of three to eight, numbers in parentheses) with at least 20 cells each. D, CCh (100 µM)-induced and TRP{gamma}-mediated Mn2+ quench of Fura-2 fluorescence was inhibited by application of Gd3+ (n = 3), La3+ (n = 3), or SKF-96365 (n = 4) (100 µM each) at the start of the experiments. Black and gray columns represent the effect of Cch on TRP{gamma}-YFP-expressing cells; the open column represents the effect of Cch on YFP-expressing cells. Bars represent the TRP{gamma}-mediated Mn2+ quench as mean values {Delta}F360 (%) 200 s after application of the blocker ± S.E. of at least three independent experiments with at least 20 cells each.

 
TRP{gamma} Forms a Receptor-regulated Cation Channel—Next, we studied TRP{gamma} in transiently transfected HEK293 cells. Fluorescence energy transfer (FRET) experiments using TRP{gamma}-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 TRP{gamma} channel complex, we initially applied Ca2+-imaging methods. In Fura-2-loaded cells, the expression of TRP{gamma} resulted in an increase in the basal Ca2+ signal (Fig. 2A), due to its spontaneous activity (17). To test whether TRP{gamma} 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 TRP{gamma}-expressing cells ~10-fold as compared with control cells (see Fig. 2A). The increase in [Ca2+]i resulted from TRP{gamma}-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 TRP{gamma}-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 TRP{gamma}-expressing cells resulted in an instantaneous slow and progressive reduction of Fura-2 fluorescence due to the spontaneous activity of TRP{gamma} (17) (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 TRP{gamma} 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 TRP{gamma}-mediated manganese quench in TRP{gamma}-expressing HEK293 cells (Fig. 2D).

In good agreement with the increased basal intracellular Ca2+ concentration, we observed spontaneous currents in whole-cell recordings from TRP{gamma}-expressing cells. Application of carbachol (100 µM) increased current responses in TRP{gamma}-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 TRP{gamma}-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 TRP{gamma} showed no clear rectification and a reversal potential (Erev) of –60 ± 20 mV (n = 10). To study the selectivity of the current of TRP{gamma} 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 TRP{gamma} were PNa/PNMDG = 1:0.15, and PCs/PNa = 1:1 (n = 6).


Figure 3
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  		FIGURE 3.
Activation of TRP{gamma}-mediated currents by carbachol. A, current-voltage relationship of whole-cell currents recorded from TRP{gamma}-YFP-expressing cells before and 1 min after the application of carbachol (100 µM). B, current-voltage relationships of whole-cell currents from TRP{gamma}-YFP-expressing cells before and 1 min after application of CCh. Currents (n = 8) were normalized for the cell capacitance. Data are means ± S.D. (• before and {circ} after application of CCh). TRP{gamma}-YFP-expressing cells were subjected to a series of voltage commands (–70 to 70 mV in 10-mV increments) in the absence (C) and presence (D) of CCh. E, time course of inward current amplitudes recorded from TRP{gamma}-expressing cells during the extracellular application of carbachol (100 µM). F, current-voltage relationships of normalized whole-cell currents recorded from TRP{gamma}-YFP-expressing cells in the presence of extracellular Na+ (•, n = 5, means ± S.D.) or NMDG ({circ}, n = 5, means ± S.D.).

 
Analysis of the Signaling Pathway Leading to TRP{gamma} Activation—To clarify whether TRP{gamma} is activated by phospholipase C-generated mediators, we used the phospholipase C inhibitor U73122. [GenBank] In the presence of U73122 [GenBank] , the application of carbachol to TRP{gamma}-transfected HEK293 cells did not result in an increase in [Ca2+]i, suggesting a phospholipase C-dependent activation of TRP{gamma} (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 TRP{gamma}-mediated Ca2+ entry in HEK293 cells transfected with TRP{gamma} DNA (Fig. 4). Carbachol reproducibly induced an increase in intracellular Ca2+ in TRP{gamma}-transfected cells, whereas OAG did not result in increases in [Ca2+]i either in TRP{gamma}-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 TRP{gamma} (Fig. 4C).

To determine the putative phospholipase C-dependent and diacylglycerol-independent activation of TRP{gamma}, 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 (23–25). 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, the four PLA2 inhibitors blocked carbachol-induced activation of TRP{gamma} with different efficacies (see Fig. 5). In summary, these data argue for a phospholipase C- and phospholipase A2-dependent activation of TRP{gamma}.

TRP{gamma} Forms a Cation Channel Regulated by Polyunsaturated Fatty Acids—Because an activation of PLA2 enzymes results in an increase in AA, we tested whether TRP{gamma}-transfected HEK293 cells can be activated by AA. The application of AA increased [Ca2+]i in Ca2+-imaging experiments and attenuated the Fura-2 fluorescence in manganese quench experiments (Fig. 6, A and B). AA is intensively metabolized by lipoxygenases, cyclooxygenases, and cytochrome P450 isoenzymes, resulting in a great variety of compounds with diverse function. To test whether the effect of AA is mediated directly or by a metabolite, we studied the effects of ETYA as a common inhibitor of AA-metabolizing enzymes (26). However, upon application of ETYA in the absence of AA, there were still responses measured (Fig. 6, C and D). This stimulating effect of ETYA was reproducibly detected in manganese quench and Ca2+-imaging experiments.

To test whether or not TRPL and TRP{gamma} are activated by different unsaturated fatty acids, we measured fluorescence changes in Fluo-4-loaded cells in a FLIPRTetra. The application of different fatty acids resulted in comparable increases in intracellular calcium in TRPL- and TRP{gamma}-expressing cells (supplemental Fig. S3).


Figure 4
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  		FIGURE 4.
TRP{gamma} is insensitive to mono- and diacylglycerols. A, effects of either 100 µM OAG or 100 µM CCh on HEK293 cells expressing TRP{gamma}-YFP. The lines represent mean values from six independent Fura-2 experiments with at least 20 cells each. Application of substances is indicated by an arrow. B, effect of either 100 µM OAG or 100 µM CCh on HEK293 cells expressing TRPC6-YFP. Shown are mean values from four independent experiments with at least 20 cells each. Application of substances is indicated by an arrow. C, effects of decanoyl-rac-glycerol (MDG, 100 µM), 1-oleoyl-rac-glycerol (MOG, 100 µM), 1,2-dioctanoyl-sn-glycerol (DOG, 100 µM), and 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 µM) on TRP{gamma}-YFP-transfected cells (dark gray filled columns), TRPC6-YFP-expressing cells (bright gray filled columns), and YFP-expressing cells (open columns). Columns represent the increases in [Ca2+]i 200 s after application of lipids as means ± S.E. of independent Fura-2 experiments (numbers given in parentheses) with at least 20 cells each.

 


Figure 5
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  		FIGURE 5.
Effects of the PLA2 inhibitors on CCh-induced TRP{gamma} stimulation. Carbachol-induced activation of TRP{gamma}-YFP-expressing HEK293 cells was studied by Mn2+ influx experiments. After a preincubation period of 5 min in the presence of the PLA2 inhibitors N-(p-amylcinnamoyl)anthranilic acid (ACA, 10 µM; n = 6) (A), bromoenol lactone (BEL, 20 µM; n = 4) (B), p-bromphenacyl bromide (pBPB, 50 µM; n = 5) (C), or 10 min in the presence of arachidonyltrifluoromethyl ketone (AACOCF3, 20 µM; n = 5) (D), carbachol (100 µM) was applied. All traces represent mean values from n independent experiments with at least 20 cells each.

 
Electrophysiological Characterization of ETYA-induced TRP{gamma} Currents—For electrophysiological characterization of the polyunsaturated fatty acid-dependent activation of TRP{gamma}, we used ETYA, the most potent activator, being an enzymatically and chemically stable AA derivative. In standard extracellular solutions, the current-voltage relationship of the ETYA-induced current, measured from voltage ramps, had an outwardly rectifying form comparable with the curves resulting from carbachol application (see Figs. 3A and 7A). Maximal currents were transiently recorded 40 s after the application of ETYA (Fig. 7B). Stable steady states were obtained 60 s after application with mean current densities of –42 ± 29 pA/pF and +114 ± 31 pA/pF (n = 12) at –90 and +90 mV, respectively (Fig. 7C). In contrast, ETYA had no effect on untransfected HEK293 cells (Fig. 7D). Enhanced inward currents and subsequent rapid desensitization were only found after application of ETYA. Note: The different current densities in carbacholand ETYA-stimulated cells may result from different stimulus intensities.

For characterization of the single channel conductance, we performed inside-out recordings from TRP{gamma}-expressing cells (n = 6) (Fig. 8). Spontaneous activity of TRP{gamma} was also recorded in the inside-out configuration showing a relatively high open probability from the start of the recordings (Fig. 8A). The application of ETYA induced an increase in channel activity within a time scale of 10 s (see Fig. 8A). Exemplary traces in the absence and presence of ETYA are shown as insets. For further quantification of our single channel data before (Fig. 8B) and after (Fig. 8C) application of ETYA, we calculated amplitude histograms of single channel amplitudes from current traces of 4-s length. The calculation revealed that ETYA increased the open probability of TRP{gamma} from 0.15 (see Fig. 8B) to 0.85 (see Fig. 8C). At the holding potential –50 mV the amplitude of the current was 8.9 pA. The chord conductance calculated from the data of our experiments is 148 pS.


Figure 6
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  		FIGURE 6.
Arachidonic acid and ETYA activate TRP{gamma}. Polyunsaturated fatty acid-induced activation in TRP{gamma}-YFP-expressing HEK293 cells was studied by Ca2+ imaging (A, C) and Mn2+ influx (B, D). Arachidonic acid (AA, 20 µM; n = 7) (A, B) and the non-metabolizable arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (ETYA, 20 µM; n = 7) (C, D) was applied to TRP{gamma}-YFP- and YFP-expressing cells. Shown are means from three independent experiments with at least 20 cells each.

 


Figure 7
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  		FIGURE 7.
Activation of TRP{gamma} by ETYA. A, whole-cell currents were recorded from TRP{gamma}-expressing cells. The application of 5,8,11,14-eicosatetraynoic acid (ETYA, 20 µM) resulted in an increase in outward and inward currents. B, time course of inward currents at a holding potential of –50 mV were recorded from TRP{gamma}-expressing cells during extracellular application of ETYA (20 µM). Normalization of currents from TRP{gamma}-expressing (C) and untransfected control cells (D) shows that ETYA selectively increased whole-cell currents in TRP{gamma}-expressing cells before (• and after {circ} application of ETYA. The data of the normalized currents were routinely taken 60 s after the application of ETYA, reflecting the stable steady state of the ETYA response.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TRP{gamma}, as a member of the TRPC channel family, has been discussed as participating in Drosophila phototransduction (12, 17). In the first report describing TRP{gamma}, its expression was shown to specifically occur in Drosophila eyes and the head, but not in the body (17), suggesting a possible sensory function of TRP{gamma}. In contrast, a second report showed that the transcription of TRP{gamma} mRNA not only occurs in the fruit fly's head but also in the cells of the Malpighian tubules (18). Similarly, our present RT-PCR data support the notion that TRP{gamma} expression is not restricted to Drosophila head. The fact that there are only three TRPC members (TRP, TRPL, TRP{gamma}) in the genome of Drosophila and the expression of TRP{gamma} in Drosophila heads and bodies make it likely that TRP{gamma}-mediated Ca2+ influx is integrated in many receptor-mediated signaling pathways as shown for the mammalian TRPC channel proteins. Indeed, TRP{gamma}, when heterologously expressed in HEK293 cells, can be regulated by hormonal activation of the cells via endogenous muscarinic GPCR, because both Ca2+-imaging and whole-cell patch clamp experiments showed an increased influx into TRP{gamma}-expressing cells.

The signaling pathway leading to TRP{gamma} activation was clarified with the help of pharmacological tools. The data in this study revealed that TRP{gamma}, like TRPL, is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A2-dependent manner. Whereas the expression of a phospholipase C isoenzyme in Drosophila has been known since 1988 (27, 28), a gene coding for a phospholipase A2 isoenzyme has been found only recently (29). Its gene product, called Radish, has been shown to be expressed in a specific subset of neurons involved in synaptic transmission necessary for olfactory memory. However, due to the lack of histological localization of TRP{gamma}, it remains unclear whether TRP{gamma} and Radish are involved in olfactory memory.


Figure 8
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  		FIGURE 8.
Fatty acid regulation of TRP{gamma} channel activity in inside-out patches. ETYA stimulated TRP{gamma} channels in inside-out patches. A, plot of NPo against time during a representative experiment shows the increase in channel activity after the application of the ETYA. Insets, currents recorded at the times indicated by (1) and (2). Channel activity was recorded in inside-out patches from HEK293 cells expressing TRP{gamma}-YFP at the holding potential of –50 mV. Statistical analysis of single channel amplitudes were calculated from current traces of 4-s length using amplitude histograms fitted to Gaussian functions before (B) and after (C) application of ETYA. Note the change in probability of channel opening from 0.15 (B) to 0.85 (C).

 
The PLA2 inhibitors used in this study have been shown to be specific for different PLA2 isoenzymes. N-(p-Amylcinnamoyl)anthranilic acid has been used as a common inhibitor, whereas p-bromphenacyl bromide, bromoenol lactone, and arachidonyltrifluoromethyl ketone have been described to be specific for the secretory, inducible, and cytosolic PLA2, respectively (30). Differences in inhibiting the carbachol-induced and TRP{gamma}- or TRPL-mediated Ca2+ influx by these PLA2 inhibitors are, therefore, likely due to their different efficacies in inhibiting the endogenously expressed PLA2 in HEK293 cells.

The concentration-response relations of PUFAs show that TRP{gamma} and TRPL are activated by polyunsaturated fatty acids with comparable efficacies, whereas palmitoleic acid, a monounsaturated fatty acid, is ineffective. Both channel proteins are stimulated not only by naturally occurring polyunsaturated fatty acids, ETYA, a synthetic PUFA analogue, also induced TRP{gamma} currents. After the application of ETYA, we obtained current-voltage relationships similar to those measured under carbachol.

TRP{gamma} in HEK293 cells was constitutively active, with a current-voltage relationship reversing around 0 mV and a permeability for cations PNa:PCs:PNMDG = 1:1:0.15, characterizing TRP{gamma} as a non-selective cation channel. This is in good agreement with earlier data of Xu et al. (17). The amplitudes of inward and outward currents increased within 30 s after the application of carbachol, indicating that TRP{gamma} is activated by mediators generated by intracellular signaling cascades. The ETYA-induced currents showed a current-voltage relationship and single channel conductance that is well comparable with that of the spontaneous currents.

The data on TRP{gamma} obtained in this study, together with those for TRPL and TRP obtained by Chyb et al. (16), demonstrate that all members of the Drosophila TRPC family are regulated by fatty acids. In terms of phylogeny of signaling cascades, the questions arise whether regulation by fatty acids is a conserved feature and can also be found in other species or whether this mechanism is specific for flies. At present, it appears that most of the mammalian TRPC channels are activated by diacylglycerols (TRPC2, TRPC3, TRPC6, and TRPC7), whereas activation by fatty acids is a still unknown principle for mammalian TRPC channels (13–15). This is in line with our study in which PUFAs did not induce current signals in mock-transfected HEK293 cells, which endogenously express TRP channels. It therefore appears unlikely that endogenous TRP channels in HEK293 cells interfere with the activation of TRP{gamma} by PUFAs.

The presence of endogenously expressed TRP channels in HEK293 cells is long known. For example, the first cloned TRP-homologous channel protein, TRPC1, has been cloned from HEK293 cells (31) as well as TRPC3 (32). In the meantime, RT-PCR experiments confirmed the expression of nearly all mammalian TRPC channels in HEK293 cells (33–35). The presence of natively expressed TRPC channels in HEK293 accounts for the receptor-induced responses recorded in mock-transfected cells and is discussed as contributing to the channel complexes formed in HEK293 cells heterologously expressing TRPC channels (36).

The work by Xu et al. (17) showed that heterologously expressed TRP{gamma} forms a functional heteromeric channel complex together with TRPL. This ability would allow TRP{gamma} and other TRP channel subunits to potentially generate a diversity of heteromeric channels, each with properties specifically tailored to a particular cellular function. This feature is a well known principle in other non-selective cation channel families, e.g. the cyclic nucleotide-gated channel subunits (37), the P2X channels (38), or the nicotinic acetylcholine channels (39). Results of fluorescence energy transfer assays and co-localization and co-immunoprecipitation studies have shown that co-expression of closely related proteins of the mammalian TRPC, TRPV, or TRPM families resulted in the formation of heteromeric channel complexes in HEK293 cells (40–42). Despite these results, co-expression of TRP channels in heterologous systems, such as HEK293 cells, had little impact on the functional properties of the channels studied. So far, only one study described that the co-expression of TRPC1 and TRPC5 resulted in a current-voltage relationship that differed from that of the homomeric channels (43). However, Nilius et al. (44) showed that the heterologous expression of TRPV4 in HEK293 resulted in the down-regulation of a TRP-unrelated channel, the natively expressed volume-regulated anion channel. Any contribution of endogenous TRP channels of HEK293 cells to the regulation of the heterologously expressed TRP{gamma} channels remains to be proven.

Whereas biochemical data argue in favor of a promiscuous interaction among TRP, TRPL, and TRP{gamma} channels (17), the analysis of fly phototransduction demonstrated that TRPL channels alone translocated from the membrane of the rhabdomeres to intracellular membrane compartments in response to light (45). The fact that TRPL and TRP{gamma} in our hands formed unitary homomeric channels activated by fatty acids may support the notion that these channels are involved in selective cellular signaling cascades, beyond their heteromeric function in phototransduction.

In summary, our data show for the first time that TRP{gamma} forms a Ca2+-permeable non-selective cation channel directly activated by polyunsaturated fatty acids generated from GPCR-dependent signaling pathways. Whereas a specific functional role of TRP{gamma} in Drosophila phototransduction appears most attractive, its body-wide expression led us to anticipate, however, a more general role of TRP{gamma} in the entire organism of the fruit fly.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie, and Sonnenfeld-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. Back

1 To whom correspondence should be addressed. Tel.: 49-30-84451825; Fax: 49-30-84451818; E-mail: Christian.Harteneck{at}charite.de.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Inge Reinsch for technical assistance, Craig Montell for providing TRP{gamma} cDNA, and Christian Grimm for help in Ca2+ imaging and fluorescence energy transfer experiments and for kindly reading the manuscript.



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TRP channels and lipids: from Drosophila to mammalian physiology
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