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. 2022 Oct 11;15(755):eabl6179.
doi: 10.1126/scisignal.abl6179. Epub 2022 Oct 11.

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels

Takaaki Sokabe  1   2   3 , Heather B Bradshaw  4 , Makoto Tominaga  2   3 , Emma Leishman  4 , Avinash Chandel  1 , Craig Montell  1
Affiliations

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels

Takaaki Sokabe et al. Sci Signal. .

Abstract

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cβ (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

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Figures

Fig. 1.
Fig. 1.. Relative lipid levels in control (w1118), norpAP24 and inaEN125 heads from flies maintained in the dark and after light exposure.
(A) Schematic of protocol for collecting heads from flies maintained at 37°C in the dark for 8 minutes or from flies kept in the dark for 3 minutes and then exposed to blue light for 5 minutes. “Dark” indicates flies that were processed using a dim photographic safety light right before the 37°C incubation, which is functionally dark to Drosophila. After freezing in liquid N2, and vortexing, the heads were collected over a sieve, lipids were extracted and analyzed by LC/MS/MS. (B) Pathway for production of endocannabinoids and other lipids from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. DAG, diacylglycerol; 2-LG, 2-linoleoyl glycerol; 2-MAG, 2-monoacylglycerol; LA, linoleic acid. (C to F) Concentrations (nmoles/gram) of the indicated lipids extracted from control and norpAP24 heads that were kept in the dark or exposed to blue light: 2-linoleoyl glycerol (2-LG) levels after 5-minute blue light exposure (C), 2-LG levels after 10-second blue light exposure (D), linoleoyl ethanolamide (LEA) levels after 5-min blue light exposure (E), and phospho-linoleoyl ethanolamide (phospho-LEA) levels after 5-min blue light exposure (F). The lipid metabolites in (C), (E) and (F) were analyzed from the same set of samples. Data are presented as means ± SEMs (n=12 independent experiments; ~1,800 fly heads per group). *p < 0.05, **p < 0.01 (Mann-Whitney U test). (G) Concentrations (nmoles/gram) of 2-LG extracted from inaEN125 heads that were kept in the dark or exposed to blue light for 5 minutes. The control maintained in dark was used for comparison. The difference in the concentration of 2-LG from (C) is due to modifications in the LC/MS/MS system. Data are presented as means ± SEMs (n=12 independent experiments; ~1,800 fly heads per group). ***p < 0.001 (Steel test).
Fig. 2.
Fig. 2.. Effects of endocannabinoids and N-acyl glycine on TRPL-dependent changes in Fura-2 ratio in S2 cells.
(A) Comparison of the maximum increase in intracellular Ca2+ (Ca2+i) in response to the indicated lipids. Lipids were added at 100 μM, except for 300 μM oleic acid (OA). The Cu2+ (−) cells did not express TRPL and the Cu2+ (+) cells expressed TRPL. Data are presented as means ± SEMs (n=3—6 independent experiments; ~300—600 cells per group). (B to F) Fura-2 dose responses of TRPL-expressing cells to 2-LG (B) and representative traces in response to 2-LG (C). Values in (B) were normalized to ionomycin (Iono). Fura-2 dose responses to 2-LG, LEA, LinGly and LA (D). Representative traces in response to 100 μM LEA (E) and to 100 μM LinGly (F) are shown. The data are the maximum Ca2+i during the stimulation period (60—360 seconds after lipid addition). Basal values were subtracted, and percentages were normalized to the maximum values obtained with 5 μM Iono. Curves were fitted using nonlinear regression with variable slopes. The red and black bars (B, C, E and F) indicate the perfusion of the lipids and Iono, respectively. Data are presented as means ± SEMs (n=5—7 independent experiments; ~500—700 cells per group).
Fig. 3.
Fig. 3.. Effects of lipase inhibitors and combination of lipids on Fura-2 responses in TRPL-expressing S2 cells.
(A) The effects of a monoacyl glycerol lipase (MAGL) inhibitor (JZL 184, 80 nM) or a MAGL/fatty acid amide hydrolase inhibitor (IDFP, 30 nM) on increases in Ca2+i induced by 2-LG (10 μM), LEA (10 μM) and LinGly (10 μM). Cells were pretreated with inhibitor or vehicle (0.1% dimethyl sulfoxide) one minute before lipid application. Background Ca2+i were obtained in non-induced Cu2+ (−) cells with the vehicle alone, 80 nM JZL 184 or 30 nM IDFP. Values were normalized to the maximum values obtained with 5 μM Iono. Data are presented as means ± SEMs (n=3 independent experiments for TRPL-expressing cells, n=4—5 independent experiments for cells not expressing TRPL. ~300—500 cells per group). (B—D) The effect of mixing 2-LG, LEA, LinGly and linoleic acid (LA) on increases in Ca2+i in TRPL-expressing cells. The numbers indicate the concentration of lipids (μM). Data are presented as means ± SEMs (n=3—4 independent experiments; ~300—400 cells per group).
Fig. 4.
Fig. 4.. Effect of 2-LG on mammalian TRPC5 and TRPC6 expressed in HEK293 cells.
(A,B) Representative Fura-2 responses to 2-LG in cells expressing mouse TRPC5 (A) or cells transfected with the empty vector (B). HEK293 cells were stimulated with 100 μM 2-LG by perfusion, then with 100 μM riluzole to check for channel function. The red and purple bars indicate the perfusion of 2-LG and riluzole, respectively. Cell viability was confirmed by applying 5 μM ionomycin (Iono, black bar). (C) Quantification of the maximum increase in intracellular Ca2+ (Ca2+i) in response to 2-LG in TRPC5-expressing cells or vector-transfected cells. (D,E) Representative Fura-2 responses to 2-LG in mouse TRPC6-expressing cells (D) and vector-transfected cells (E). HEK293 cells were stimulated with 100 μM 2-LG by perfusion, then with 100 μM GSK1702934A (GSK) to check for channel function. The red and blue bars indicate the perfusion of 2-LG and GSK1702934A, respectively. Cell viability was confirmed by applying 5 μM ionomycin (Iono, black bar). (F) Quantification of the maximum increase in intracellular Ca2+ (Ca2+i) in response to 2-LG in TRPC6-expressing cells or vector-transfected cells. The data in (C) and (F) are the maximum Ca2+i during the stimulation period (60—360 seconds after lipid addition). Basal values were subtracted, and percentages were normalized to the maximum values obtained with 5 μM Iono. Data are presented as means ± SEMs (n=13 independent experiments for TRPC5-expressing cells, n=11 independent experiments for mock-transfected cells; n=11 independent experiments for TRPC6-expressing cells, n=8 independent experiments for mock-transfected cells. ~500—800 cells per group). **p < 0.01 and ***p < 0.001 (Mann-Whitney U test).
Fig. 5.
Fig. 5.. Monitoring responses of photoreceptor cells stimulated with endocannabinoids and N-acyl glycine with GCaMP6f.
(A, B) Representative GCaMP6f responses to 2-LG in control (norpAP24) ommatidia (A) and in norpAP24;trpl302;trp343 ommatidia (B). The ommatidia were stimulated with 30 μM 2-LG, then with 5 μM ionomycin (Iono) to confirm GCaMP6f responsiveness in photoreceptor cells. The changes in fluorescence (gray scale levels 0—255) are shown in pseudo colors. The dotted lines in the images for basal conditions (before addition of 2-LG) outline individual rhabdomeres. 2-LG images were obtained at the 300 second time points in (C) and (D). Scale bars, 20 μm. (C—F) Traces showing representative Ca2+i responses (ΔF/F0) in photoreceptor cells from control (norpAP24) (C). norpAP24;trpl302;trp343 (D), norpAP24;+;trp343 (E) norpAP24;trpl302;+ (F) flies. The red and black bars indicate application of 30 μM 2-LG (60—300 seconds) and 5 μM ionomycin (after 300 seconds), respectively. (G) ΔF/F0 indicates the maximum Ca2+i responses during the stimulation period (60—300 seconds) in (C) to (F) divided by basal fluorescence levels. (H) Quantification of area under curve during the stimulation period (60—300 seconds) in (C) to (F). Data are presented as means ± SEMs in G and H. n=9—11 independent experiments; 186—292 ommatidia per group. **p < 0.01. Kruskal-Wallis with Steel post hoc test. (I) Proportion of no or low responding photoreceptor cells (max ΔF/F0 ≤0.2) during the stimulation period in (C) to (F). Data are presented as means ± SEMs. n=9—11 independent experiments; 186—292 ommatidia per group. **p < 0.01, ***p < 0.001. One-way ANOVA with Tukey’s post hoc analysis. (J) Traces showing representative Ca2+i responses (ΔF/F0) in control (norpAP24) photoreceptor cells in a bath containing 1.5 mM Ca2+ (blue in the bar near the top) or no Ca2+ (white in the bar near the top). The red and black bars indicate application of 30 μM 2-LG and 5 μM Iono, respectively. The dotted and solid orange lines indicate ΔF/F0 in a Ca2+-containing bath in the absence (dotted) and presence (solid) of 2-LG. The dotted and solid green lines indicate ΔF/F0 in a bath without added Ca2+ in the absence (dotted) and presence (solid) of 2-LG. (K) Quantification of the maximum Ca2+i responses to 30 μM 2-LG in the absence (−) or the presence (+) of 1.5 mM extracellular Ca2+. ΔF/F0 in each Ca2+ condition was calculated using values in the periods indicated by green and orange dotted and solid lines in (J). Data are presented as means ± SEMs. n=8 independent experiments. Total: 211 ommatidia. ***p < 0.001. Paired Student’s t-test. (L) Quantification of the maximum Ca2+i responses to 30 μM 2-LG in the presence (+) or absence (−) of 10 μM ruthenium red (RuR). Maximum Ca2+i responses in the presence of RuR (first 2 minutes) and after RuR washout (the following 3 minutes). Data are presented as means ± SEMs. n=9 independent experiments. Total: 238 ommatidia. ***p < 0.001. Paired Student’s t-test. (M, N) Maximum Ca2+i responses as measured by the GCaMP6f reporter of control (norpAP24) and norpAP24;trpl302;trp343 photoreceptor cells to 100 μM LEA or 30 μM LinGly during the stimulation period (4 minutes). In the absence of the one outlier for LinGly, the significance is essentially unchanged (p=0.0097). Data are presented as means ± SEMs. n=9—11 independent experiments. 175—325 ommatidia per group. **p < 0.01, ***p < 0.001. Unpaired Student’s t-test for LEA and Mann-Whitney U test for LinGly.
Fig. 6
Fig. 6. Monitoring GCaMP6f responses of photoreceptor cells stimulated with 2-LG and osmotic solutions.
(A, B) Representative GCaMP6f responses to a hypotonic (~216 mOsm) solution in control (norpAP24) ommatidia (A) and norpAP24;trpl302;trp343 ommatidia (B). (C) ΔF/F0 indicates the maximum Ca2+i responses during the stimulation period (60—300 seconds) in (A) and (B) divided by basal fluorescence levels. Data are presented as means ± SEMs. control, n=11 independent experiments, 302 total ommatidia; norpAP24;trpl302;trp343, n=9 independent experiments, 202 total ommatidia. *p < 0.05. Unpaired Student’s t-test. (D) Representative GCaMP6f responses to 2-LG in control ommatidia (norpAP24) followed by a combination of 2-LG and a hypotonic solution. The red and blue bars indicate application of 30 μM 2-LG (60—300 seconds) and the hypotonic solution (~216 mOsm) (after 180 seconds), respectively. (E) Quantification of the maximum Ca2+i responses of control ommatidia (norpAP24) to a hypotonic solution alone, 30 μM 2-LG alone, or a combination of a hypotonic solution and 2-LG. Data are presented as means ± SEMs. n=11—12 independent experiments. 274—358 ommatidia per group. *p < 0.05, ***p < 0.001. One-way ANOVA with Tukey’s post hoc analysis. (F) Representative GCaMP6f responses to 2-LG followed by a combination of 2-LG and hypertonic solution in control (norpAP24)ommatidia. The red and magenta bars indicate application of 30 μM 2-LG (60—300 seconds) and hypertonic solution (~416 mOsm; after 180 seconds), respectively.
Fig. 7
Fig. 7. Endocannabinoid 2-LG activates TRP and TRPL in concert with mechanical stimulation.
Shown is the phototransduction cascade. Light (thunderbolt) activates rhodopsin, which in turn leads to exchange of GTP for GDP and activation of Gαq. Gαq-GTP activates PLC (NORPA), catalyzing the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2), and production of diacylglycerol (DAG), inositol trisphosphate (IP3) and a proton (H+). DAG has a smaller head group than PIP2, causing a conformational change in the membrane (membrane deformation), which is proposed to mechanically activate TRP and TRPL (27). Production of 2-linoleoyl glycerol (2-LG) from DAG is catalyzed by a DAG lipase (DAGL) encoded by inaE (38). We propose that TRP and TRPL are activated through dual mechanisms: by direct binding of 2-LG to the channels and by membrane deformation resulting from conversion of PIP2 to DAG.
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