gsk1702934a  (Tocris)

Journal: Nature Communications

Article Title: Structural mechanism of the agonist binding on human TRPC3 channel

doi: 10.1038/s41467-025-64435-6

Figure Lengend Snippet: a , b Cryo-EM density maps of GSK1702934A-bound state hTRPC3 shown in side view ( a ) and top view ( b ). Subunits A, B, C, and D are colored in yellow, blue, pink, and green, respectively. Ligand GSK1702934A is colored in gold. The approximate boundary of the cell membrane is indicated by gray lines. TMD transmembrane domain, ICD intracellular cytosolic domain. c Densities of GSK1702934A and nearby residues shown in side view. Densities are shown as grey surfaces. GSK1702934A is shown as sticks, and hTRPC3 is shown as cartoon. Both are colored the same as in ( a ). d , e Close-up view of the GSK1702934A-binding site. The GSK1702934A molecule and the side chains of its interacting residues are shown as sticks. f Cartoon representation of the interactions between GSK1702934A and hTRPC3. S6 of subunit A and S5, S6, pore helices of subunit B are represented as yellow ovals and blue ovals, respectively. Residues that interact with GSK1702934A are labeled inside the ovals. g Sequence alignment among TRPC1/4/5 and TRPC3/6/7 is shown. The residues that interact with GSK1702934A are marked by gold asterisks. Conserved residues are colored in gold. The corresponding domains are labeled above the sequence.

Article Snippet: The concentration of Carbachol (CCh, HarveyBio, C33526 ), 4n (Dizal Pharmaceutical), GSK1702934A (Shanghai Universal Biotech, 6508), 1-Oleoyl-2-acetyl-sn-glycerol (OAG, Sigma, O6754), 1-Oleoyl-rac-glycerol (1-MOG, Sigma, M7765), 2-Oleoylglycerol (2-MOG, TargetMol, T37526 ), BTDM (Dizal Pharmaceutical), and (-)-Englerin A (EA, Sigma, PHL82530 ) used in patch-clamp are marked out in the figures.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay, Labeling, Sequencing

Journal: Nature Communications

Article Title: Structural mechanism of the agonist binding on human TRPC3 channel

doi: 10.1038/s41467-025-64435-6

Figure Lengend Snippet: a Close-up view of DAG at the L2 site. The mutated amino acids on hTRPC3 are shown as sticks. DAG is colored in green. TRPC3 is colored in blue. b The ratio of peak currents activated by CCh and GSK1702934A in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation method for the activation current is described in detail in Supplementary Fig. . The number of biological replicates in each group is indicated below the genotype. The data are shown as the mean ± SD. The data were analyzed using Brown–Forsythe and Welch ANOVA tests, and the multiple comparisons between each group were analyzed using Dunnett’s T3 multiple comparisons tests with p -values indicated above the corresponding groups. Source data are provided as a Source Data file. c The ratio of peak currents activated by OAG and GSK1702934A in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. d The ratio of peak currents activated by CCh and 4n in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. e The ratio of peak currents activated by OAG and 4n in wild-type, E603A, K607A, and V637A under whole-cell mode at −60 mV. The calculation, annotation, and statistical analyses were identical to those described in ( b ). Source data are provided as a Source Data file. f Chemical structures of DAG, OAG, 1-MOG, and 2-MOG. g – i . Macroscopic currents of wild-type hTRPC3/6/7 recorded in the whole-cell mode. Zero current is indicated by a dashed line. The duration of ligand application is indicated by a solid line above. Source data are provided as a Source Data file.

Article Snippet: The concentration of Carbachol (CCh, HarveyBio, C33526 ), 4n (Dizal Pharmaceutical), GSK1702934A (Shanghai Universal Biotech, 6508), 1-Oleoyl-2-acetyl-sn-glycerol (OAG, Sigma, O6754), 1-Oleoyl-rac-glycerol (1-MOG, Sigma, M7765), 2-Oleoylglycerol (2-MOG, TargetMol, T37526 ), BTDM (Dizal Pharmaceutical), and (-)-Englerin A (EA, Sigma, PHL82530 ) used in patch-clamp are marked out in the figures.

Techniques: Activation Assay

Journal: Nucleic Acids Research

Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

doi: 10.1093/nar/gkae245

Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

Article Snippet: The following TRPC3/C6/C7 activators were employed in the current study: L687 ( , WO/2022/118966) , Cannabidiol (CBD) (#Axon1234; Axon Medchem, VA, USA), and GSK1702934A (#6508; Tocris Bioscience, Bristol, UK).

Techniques: Knockdown, Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

Journal: Journal of Extracellular Biology

Article Title: TRPC3 signalling contributes to the biogenesis of extracellular vesicles

doi: 10.1002/jex2.132

Figure Lengend Snippet: TRPC3 activation increases calcium signalling and EV release in SKOV3 cells. (a) Western blot of endogenous TRPC3 and β‐actin protein expression in five ovarian cancer cell lines: SKOV3, OVCAR3, OVCAR5, IGROV1 and A2780. Representative image of three independent experiments. (b) Schematic representation of NFAT translocation assay. Activation of TRPC3 stimulates a rise in intracellular calcium (Ca 2+ ) which subsequently activates the phosphatase calcineurin, inducing dephosphorylation and nuclear translocation of NFAT which can be visualized by a GFP tag. (c) Representative images of NFAT translocation in SKOV3 cells expressing GFP‐tagged NFAT following treatment with 1.25 μM ionomycin or 0.1/0.3/1/10 μM GSK1702934A, alongside untreated (‐) and DMSO controls. Fixed cells were subjected to indirect immunofluorescence using an anti‐GFP antibody and the DNA dye DAPI. Nuclear regions are indicated. Scale bars 50 μm. (d) Nuclear/cytosol fluorescence intensity ratio of treated cells shown in (c) (mean ± SEM of ≥100 cells over three independent experiments; one‐way ANOVA followed by Tukey's multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). (e) MACSPlex exosome assay data showing the mean signal intensities of CD63, CD9, CD81 and two isotype controls detected on EVs isolated from SKOV3 cells treated with 0.3 μM GSK1702934A or DMSO equivalent. APC fluorescence intensity adjusted to PBS control. Mean ± SEM of three independent isolations. (f) NTA analysis of EVs isolated from SKOV3 cells treated with 0.3 μM GSK1702934A or DMSO equivalent (mean ± SEM of three independent isolations; two‐way ANOVA followed by Tukey's multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01).

Article Snippet: The selective TRPC3 agonist GSK1702934A (Xu et al., ) (Tocris) and TRPC3 antagonist PYR3 (Kiyonaka et al., ) (Sigma‐Aldrich®) were solubilized in DMSO, and serial‐diluted in the appropriate medium to concentrations of 0.1‐10 μM at the time of cell stimulation.

Techniques: Activation Assay, Western Blot, Expressing, Translocation Assay, De-Phosphorylation Assay, Immunofluorescence, Fluorescence, Isolation, Control

Journal: Journal of Extracellular Biology

Article Title: TRPC3 signalling contributes to the biogenesis of extracellular vesicles

doi: 10.1002/jex2.132

Figure Lengend Snippet: TRPC3 activation stimulates distinct EV release. (a) Projection of total CD81‐pHlourin events in DMSO control and treated cells over a 5‐min time course. Top row, left to right: DMSO control and 1 μM PYR3. Bottom row, left to right: 0.1, 0.3 and 1 μM TRPC3 activator GSK1702934A (GSK). Pseudocoloured. Scale bars 10 μm. (b) Quantity of CD81‐pHluorin events captured in DMSO control and treated cells represented in (a) over a 5‐min time course (mean ± SEM; one‐way ANOVA followed by Tukey's multiple comparisons test; ** p ≤0.01). See Video . (c‐d) The width (c) and mean fluorescence (d) of CD81‐pHluorin events in DMSO control cells, and cells treated with 1 μM PYR3 or 0.1, 0.3, 1 μM GSK1702934A (violin plots show median and upper and lower quartiles; Kruskal‐Wallis followed by Dunn's multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). (e) Percentage of stay, fade and flash event types in DMSO control and treated cells in (a) over a 5‐min time‐course (mean ± SEM; two‐way ANOVA followed by Tukey's multiple comparisons test; ** p ≤ 0.01). (b‐e) represent a total of n≥6 cells imaged over three independent experiments.

Article Snippet: The selective TRPC3 agonist GSK1702934A (Xu et al., ) (Tocris) and TRPC3 antagonist PYR3 (Kiyonaka et al., ) (Sigma‐Aldrich®) were solubilized in DMSO, and serial‐diluted in the appropriate medium to concentrations of 0.1‐10 μM at the time of cell stimulation.

Techniques: Activation Assay, Control, Fluorescence

Journal: Journal of Extracellular Biology

Article Title: TRPC3 signalling contributes to the biogenesis of extracellular vesicles

doi: 10.1002/jex2.132

Figure Lengend Snippet: EV release in SKOV3 cells is localized and synchronized. (a) Representative images demonstrating instances of localized CD81‐pHluorin events in SKOV3 cells, indicated by white arrows. Scale bars 2 μm. (b‐c) Percentage of localized CD81‐pHluorin events (defined as events occurring within 5 μm (<5 μm) of another event in the same cell) in (b) untreated, ionomycin‐ and histamine‐treated cells and (c) untreated (‐), DMSO, PYR3 or 0.1, 0.3, 1 μM GSK1702934A‐treated cells (mean ± SEM; a total of n ≥ 6 cells imaged over three independent experiments; two‐way ANOVA followed by Tukey's multiple comparisons test; ** p ≤ 0.01, *** p ≤ 0.001). (d) Percentage of synchronized events, which occur within 5 μm (<5 μm) or further away than 5 μm (>5 μm) of another CD81‐pHluorin event in the same cell (mean ± SEM of all imaged cells regardless of treatment condition, n = 101; Wilcoxon matched‐pairs signed rank test; *** p ≤ 0.001).

Article Snippet: The selective TRPC3 agonist GSK1702934A (Xu et al., ) (Tocris) and TRPC3 antagonist PYR3 (Kiyonaka et al., ) (Sigma‐Aldrich®) were solubilized in DMSO, and serial‐diluted in the appropriate medium to concentrations of 0.1‐10 μM at the time of cell stimulation.

Techniques:

Journal: Journal of Extracellular Biology

Article Title: TRPC3 signalling contributes to the biogenesis of extracellular vesicles

doi: 10.1002/jex2.132

Figure Lengend Snippet: TRPC3 inhibition attenuates histamine‐stimulated calcium entry and EV release. (a) Representative images of NFAT translocation in SKOV3 cells expressing GFP‐tagged NFAT following treatment with 100 μM histamine or 100 μM histamine plus 3 μM PYR3, alongside untreated (‐) and DMSO controls. Fixed cells were subjected to indirect immunofluorescence using an anti‐GFP antibody and the DNA dye DAPI. Nuclear regions are indicated. Scale bars 50 μm. (b) Nuclear/cytosol fluorescence intensity ratio of treated cells shown in (a) (mean ± SEM of ≥100 cells over three independent experiments; one‐way ANOVA followed by Tukey's multiple comparisons test; ****p ≤ 0.0001). (c) Projection of total fusion events in untreated cells (top left) and cells treated with 100 μM histamine (top right), 100 μM histamine + DMSO (bottom left) or 100 μM histamine + 0.3 μM PYR3 (bottom right) over a 5‐min time course. Pseudocoloured. Scale bars 10 μm. (d) Quantity of CD81‐pHluorin events captured in untreated cells, and cells treated with histamine, histamine + DMSO and histamine + PYR3 represented in (C) over a 5‐min time course (mean ± SD; n ≥ 10 cells over two independent experiments; one‐way ANOVA followed by Tukey's multiple comparisons test; * p ≤ 0.05).

Article Snippet: The selective TRPC3 agonist GSK1702934A (Xu et al., ) (Tocris) and TRPC3 antagonist PYR3 (Kiyonaka et al., ) (Sigma‐Aldrich®) were solubilized in DMSO, and serial‐diluted in the appropriate medium to concentrations of 0.1‐10 μM at the time of cell stimulation.

Techniques: Inhibition, Translocation Assay, Expressing, Immunofluorescence, Fluorescence

Journal: Journal of Extracellular Biology

Article Title: TRPC3 signalling contributes to the biogenesis of extracellular vesicles

doi: 10.1002/jex2.132

Figure Lengend Snippet: TRPC3 activation and TRPC3 activator‐induced EVs drive SKOV3 cell growth. (a) Growth curves representing total cell number at 24, 48 and 72 h incubation with TRPC3 activator GSK1702934A (0.3 μM), TRPC3 inhibitor PYR3 (3 μM) or DMSO control (mean ± SEM; n = 4; two‐way ANOVA followed by Tukey's multiple comparisons test; * p ≤ 0.05, *** p ≤ 0.001). (b) Experimental procedure to assess TRPC3‐mediated EV communication: SKOV3 cells were grown to ∼80% confluency in complete media, washed and treated with 0.3 μM TRPC3 activator, 3 μM TRPC3 inhibitor or DMSO equivalent in serum‐free media (SFM) for 4 h. EVs were then isolated from the conditioned media and quantified by NTA. 1× and 10× EV/cell concentrations were calculated from the untreated control and added to naïve cells in media supplemented with 10% EV‐depleted FBS. Equivalent volumes of PBS were added to cells as a no EV control. Cell counts with Trypan Blue were used to assess the effects of TRPC3‐mediated EV communication. (c‐d) The relative difference in total cell number at 48 h between all treatment groups and DMSO‐treated control EVs at 1× and 10× EV treatment concentrations (mean ± SEM; n = 3; one‐way ANOVA followed by Tukey's multiple comparisons test; * p ≤ 0.05).

Article Snippet: The selective TRPC3 agonist GSK1702934A (Xu et al., ) (Tocris) and TRPC3 antagonist PYR3 (Kiyonaka et al., ) (Sigma‐Aldrich®) were solubilized in DMSO, and serial‐diluted in the appropriate medium to concentrations of 0.1‐10 μM at the time of cell stimulation.

Techniques: Activation Assay, Incubation, Control, Isolation