trpc3  (Alomone Labs)

Journal: iScience

Article Title: The role of arachidonic acid metabolites in the subtype classification and pathogenesis of primary aldosteronism

doi: 10.1016/j.isci.2025.114598

Figure Lengend Snippet: Arachidonic acid promotes Ca 2+ uptake and increases CYP11B2 expression in adrenal cortical cells (A) Confirmation of the expression of CYP11B2 in primary adrenal cortical cells. Scale bars, 50 μm. (B) Cell viability of primary adrenal cortical cells treated with different doses of arachidonic acid (AA) ( n = 4). (C) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of AA ( n = 6). (D) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of Ang II (left) or endothelin-1 (right) along with AA ( n = 3). (E) Representative western blots showing levels of CYP11B2, CYP11B1, CYP17A1, and HSD3B2 in primary adrenal cortical cells treated with vehicle or AA. The quantitative results are shown on the right ( n = 3). (F) Changes of cytoplasmic Ca 2+ , labeled with Fura-2 AM, in primary adrenal cortical cells treated with 1 mM thapsigargin (TG) stimulation in a 1 mM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (G) Changes of mitochondrial Ca 2+ , labeled with Rhod-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with 200 μM ATP stimulation in a 300 μM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (H) Changes of endoplasmic reticulum (ER) Ca 2+ , labeled with Mag Fura-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with ATP stimulation in a Ca 2+ -free extracellular solution after preincubation with vehicle or AA ( n = 12). (I) Representative western blots showing levels of KCNJ5, Na + /K + ATPase alpha-1 subunit, NCX-1, Letm 1, MCU, VDAC, RyR2, and IP 3 R in primary adrenal cortical cells treated with vehicle or AA. (Left) Representative western blots showing levels of TRPC1, TRPC3, TRPC6, TRPV1, and TRPV4 in primary adrenal cortical cells treated with vehicle or AA. (Right) The quantitative results are shown on the left ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with 1 μM AA group by one-way ANOVA (B and C) and by Student’s t test (D–I).

Article Snippet: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Expressing, Western Blot, Labeling

Journal: iScience

Article Title: The role of arachidonic acid metabolites in the subtype classification and pathogenesis of primary aldosteronism

doi: 10.1016/j.isci.2025.114598

Figure Lengend Snippet: The expression of TRPC3 in adrenal gland is reduced by salt loading (A) Representative image of hematoxylin and eosin (H&E) staining (left) and immunofluorescence staining (right) for CYP11B2 (red) and TRPC3 (green) in peritumoral adjacent tissue (PAT, top) and aldosterone-producing adenoma (APA, bottom) section. Nuclei were labeled with DAPI (blue). Scale bars, 50 μm. (B) The amount of 24-h urine output (left), 24-h urinary concentrations of electrolytes (middle), and serum concentrations of aldosterone (right) in mice treated with normal-salt diet (NSD) and high-salt diet (HSD) ( n = 6). (C) The amount of 24-h urine output (left), 24-h urinary concentrations of electrolytes (middle), and serum concentrations of aldosterone (right) in rats treated with NSD and HSD ( n = 6). (D) Representative images of H&E staining (left), immunohistochemical staining for CYP11B2 (middle), and immunofluorescence staining for TRPC3 (red, right) in adrenal gland sections from mice treated with NSD and HSD for 2 weeks. Scale bars, 50 μm. (E) Representative images of H&E staining (left), immunohistochemical staining for CYP11B2 (middle) and immunofluorescence staining for TRPC3 (red, right) in adrenal gland sections from rats treated with NSD and HSD for 2 weeks. Scale bars, 50 μm. (F) Representative western blots showing levels of TRPC3 and CYP11B2 in the adrenal gland (left). The quantitative results are shown in the middle (mouse) and on the right (rat) ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with NSD group by Student’s t test (B–F). C, capsule; ZG, zona glomerulosa; ZF, zona fasciculata.

Article Snippet: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Expressing, Staining, Immunofluorescence, Labeling, Immunohistochemical staining, Western Blot

Journal: iScience

Article Title: The role of arachidonic acid metabolites in the subtype classification and pathogenesis of primary aldosteronism

doi: 10.1016/j.isci.2025.114598

Figure Lengend Snippet: Arachidonic acid promotes proliferation, migration, and aldosterone secretion of adrenal cortical cell in a TRPC3-dependent manner (A) Changes of cytoplasmic Ca 2+ , labeled with Fura-2 AM, in primary adrenal cortical cells treated with 1 mM thapsigargin (TG) stimulation in a 1 mM extracellular Ca 2+ solution after preincubation with vehicle, arachidonic acid (AA), pyr3, and/or GSK1702934A ( n = 12). (B) Changes of mitochondrial Ca 2+ , labeled with Rhod-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with 200 μmol/L ATP stimulation in a 300 μM extracellular Ca 2+ solution after preincubation with vehicle, AA, pyr3, and/or AA + GSK1702934A ( n = 12). (C) Changes of endoplasmic reticulum Ca 2+ , labeled with Mag Fura-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with ATP stimulation in a Ca 2+ -free extracellular solution after preincubation with vehicle, AA, pyr3, and/or GSK1702934A ( n = 12). (D) Representative images of Hochest 33342 (blue) and EdU staining (red) (top), transwell migration assay (middle), and wound healing assay (bottom) in primary adrenal cortical cells treated with vehicle, AA, pyr3, and/or GSK1702934A. The quantification percentage of EdU-positive cells, migrated cell numbers measured by transwell migration assay, and migration area measured by scratch wound healing assay are shown on the right ( n = 6). (E) Levels of aldosterone in the cellular supernatant of primary adrenal cortical cells treated with AA and/or short hairpin (sh)-TRPC3 ( n = 6). (F) Representative western blots showing levels of TRPC3 and CYP11B2 in primary adrenal cortical cells treated with AA and/or sh-TRPC3. The quantitative results are shown on the right ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with the vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with the AA group by one-way ANOVA (A–F).

Article Snippet: TRPC3 , Alomone , Cat#ACC-016; RRID: AB_2040236.

Techniques: Migration, Labeling, Staining, Transwell Migration Assay, Wound Healing Assay, Western Blot

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) Schematic of one TRPC3 subunit with sites of regulation and modification highlighted. AR, ankyrin repeat; CTD, C-terminal domain; CIRB, calmodulin/IP 3 R-binding domain; L, linker helix; NAG, N-acetylglucosamine; TRP, transient receptor potential domain; VSLD, voltage-sensing-like domain. The Moonwalker (Mwk) mutation (T561A) and SCA41 patient variant (R677H) both confer pathogenic gain of function to the channel. (B) TRPC3 subunit structure (PDB code 6CUD) with the plasma membrane indicated, labelled as in (A).

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Modification, Binding Assay, Mutagenesis, Variant Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) 2D chemical structure of TRPC3 inhibitor GSK2820986A (GSK-986). Drawing generated using ChemSketch. (B) pIC50 values (-logIC 50 ) for GSK-986 obtained from high-throughput screening using FLIPR assay and automated planar perforated patch-clamp electrophysiology (Ion Works Quattro). (C) Dose-response curve for GSK-986 inhibition of TRPC3 expressed in HEK293T cells. Whole-cell patch-clamp recordings following activation with 1μM GSK1702934A in the presence of GSK-986. IC 50 =0.08 nM (n=5 cells). (D) Representative whole-cell currents recorded from HEK293T cells transiently transfected with TRPC3, activated using 1 μM GSK1702934A (GSK 170 ) and exposed to 0.03-300 nM GSK-986 or DMSO control. (E) Schematic of the GFP-NFAT translocation assay. Upon activation of TRPC3 by GSK1702934A and subsequent increase of cytoplasmic calcium concentration, calcium binds and activates calmodulin (CaM), which phosphorylates and activates the phosphatase calcineurin (CaN). Phosphorylated CaN dephosphorylates GFP-tagged NFAT, which translocates to the nucleus. Figure created in Biorender. (F) GSK-986 significantly reduces nuclear GFP-NFAT localization in Neuro-2a cells transiently co-transfected with wildtype (WT) FLAG-TRPC3 and GFP-NFAT compared to DMSO vehicle-treated control. Cells were treated with 10 μM of the activator GSK1702934A and either 1 μM GSK-986 or an equivalent volume of DMSO. The percentage of nuclear-localized GFP-NFAT represents TRPC3 activity. Unpaired two-tailed T-test, **p<0.01, n=3 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± standard deviation (SD). (G) Representative images of NFAT-GFP translocation experiments quantified in (F). Treated cells were fixed and immunostained with antibodies against FLAG and GFP. Nuclei are visualized with DAPI in blue. Image windows are expanded to show NFAT localization in individual cells. Scale bars: 50 μm. (H) GSK-986 significantly reduces nuclear NFAT localization in Neuro-2a cells transiently transfected with TRPC3 harboring the SCA41 GOF disease mutation p.R677H. Cells were treated with either 1 μM GSK-986 or an equivalent volume of DMSO. The percentage of nuclear-localized GFP-NFAT represents TRPC3 activity. Unpaired two-tailed T-test, ***p<0.001, n=4 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± SD. (I) Representative images of NFAT-GFP translocation experiments quantified in (H). Treated cells were fixed and immunostained with antibodies against FLAG and GFP. Nuclei are visualized with DAPI in blue. Image windows are expanded to show NFAT localization in individual cells. Scale bars: 50 μm.

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Generated, High Throughput Screening Assay, Patch Clamp, Inhibition, Activation Assay, Transfection, Control, Translocation Assay, Concentration Assay, Activity Assay, Two Tailed Test, Standard Deviation, Mutagenesis

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) TRPC3 structure PDB 6CUD embedded in a bilayer of the model lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphocholin (POPC). (B) Top 20 results from Autodock Vina docking of GSK-986 against the whole protein from the final frame of 100-ns TRPC3 simulation (6CUD), with ligands shown as spheres. (C) Top-down (extracellular) view of top 20 docking poses. Three out of four S4/5 pockets are occupied by GSK-986. (D) Top 20 results from focused docking of GSK-986 against the S4-S5 pocket. (E) Pose chosen for further simulation. (F) The stable GSK-986 pose in the S4-S5 pocket, following equilibration, is in close contact to the sidechain of residue Q555, and the backbone of V535. (G) The pathogenic GOF mutations SCA41 (R677H) and Mwk (T561A) are also located in the S4-S5 region. (H) The predicted GSK-986 binding pose is analogous to the structurally resolved pose of the non-selective TRPC inhibitor BTDM in TRPC6 (cyan, PDB 7DXF). The equivalent glutamine residue in the S4-S5 linker in this structure is highlighted (cyan).

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) No significant difference in maximum and outward current density values was recorded using whole-cell patch-clamp between HEK293T cells transfected with TRPC3 wildtype (WT) and Q555A mutation constructs under both basal conditions and activation with 10 μM GSK1702934A (GSK170). Two-way ANOVA with Šídák’s multiple correction, **p=0.0041, n=5 cells. Scatter points represent biological replicates. Current density measurements were taken at 100mV, adjusted for baseline at a 0 mV hold. (B, C) Representative TRPC3 currents recorded from transiently transfected HEK293T cells using whole-cell patch-clamp. Cells were transfected with either WT TRPC3 (B) or the TRPC3 Q555A mutation construct (C). Basal traces were taken before activation with 10 μM GSK1702934A to compare with traces at peak activation, prior to desensitization. (D) Mutation Q555A significantly impairs the inhibition of TRPC3 by GSK-986 as demonstrated by impaired nuclear NFAT-GFP translocation in Neuro-2a cells. Cells were transiently transfected with WT or Q555A mutant FLAG-TRPC3 and treated with 10 μM of the activator GSK1702934A and either 1 μM GSK- 986 or an equivalent volume of DMSO. The percentage of nuclear GFP-NFAT of DMSO-treated cells was used as the maximum activation of TRPC3 and compared to inhibitor-treated cells. Two-way ANOVA followed by Šídák’s multiple comparisons test, **p=0.0041, n=5 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± standard deviation (SD). (E) Representative images of NFAT-GFP translocation experiments quantified in (D). Treated cells were fixed and immunostained with antibodies against FLAG and GFP. Nuclei are visualized with DAPI in blue. Scale bars: 50µm. Image windows are expanded to show NFAT localization in individual cells. (F) The Q555A mutation significantly impairs the GSK-986-mediated inhibition of TRPC3 harboring the GOF disease mutation R677H, as demonstrated by impaired nuclear NFAT-GFP translocation in Neuro-2a cells. Cells were transfected with FLAG-TRPC3 R677H with and without the additional Q555A mutation and treated with either 1 μM GSK-986 or an equivalent volume of DMSO. The percentage of nuclear GFP-NFAT of DMSO-treated cells was used as the maximum activation of TRPC3 and compared to inhibitor-treated cells. Two-way ANOVA followed by Šídák’s multiple comparisons test, **p=0.005, n=3 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± SD. (G) Representative images of NFAT-GFP translocation experiments quantified in (H). Treated cells were fixed and immunostained with antibodies against FLAG and GFP. Nuclei are visualized with DAPI in blue. Scale bars: 50µm. Image windows are expanded to show NFAT localization in individual cells.

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Patch Clamp, Transfection, Mutagenesis, Construct, Activation Assay, Inhibition, Translocation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) GSK-986 (lilac mesh) in its stable interaction pose in the TRPC3 S4-S5 binding pocket, with close-contact residues highlighted in orange. (B) Five residues of interest form close contact with GSK-986. (C) NFAT-GFP translocation assay in Neuro-2a cells transfected with FLAG-TRPC3 harboring the GOF disease mutation R677H and additional alanine substitutions in residues of interest. Cells were treated with 1 μM GSK-986 inhibitor. Introduction of the L558A mutation significantly impairs the inhibition of NFAT-GFP nuclear translocation in GSK-986-treated cells. The percentage of nuclear GFP-NFAT of DMSO-treated cells was used as the maximum activation for each TRPC3 construct and to which inhibitor-treated cells were compared. Ordinary one-way ANOVA followed by Holm-Šídák’s multiple comparisons test, ****p<0.0001, n=3 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± standard deviation (SD). (D) High amino acid sequence conservation between TRPC3 and TRPC6 across the helices enclosing the S4-S5 pocket. (E) GSK-986 (lilac mesh) within the S4-S5 pocket with TRPC3 residues that differ in TRPC6 highlighted in orange. (F) Residues L571, F577, I585, I641, and I648 differ between the TRPC3 and TRPC6 sequence and are in close proximity to the GSK-986 binding site in the S4-S5 pocket. (G) NFAT-GFP translocation assay in Neuro-2a cells transfected with FLAG-TRPC3 harboring the GOF disease mutation R677H and additional TRPC3-to-TRPC6 substitutions in residues of interest. Cells were treated with 1 μM GSK-986 inhibitor. Introduction of the F577V mutation significantly impairs the inhibition of NFAT-GFP nuclear translocation in GSK-986-treated cells. The percentage of nuclear GFP-NFAT of DMSO-treated cells was used as the maximum activation for each TRPC3 construct and to which inhibitor-treated cells were compared. Lognormal ordinary one-way ANOVA followed by Holm-Šídák’s multiple comparisons test, **p=0.002, n=3 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± SD. (H, I) The TRPC3 F577 residue may have different functional effects on the channel compared to the equivalent valine residue in TRPC6. TRPC3 F557 appears to engage in perpendicular π-stacking with F538 on the adjacent S4 helix (H), while the TRPC6 V646 cannot (I).

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Binding Assay, Translocation Assay, Transfection, Mutagenesis, Inhibition, Activation Assay, Construct, Standard Deviation, Sequencing, Residue, Functional Assay

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A, B) Alignments between the GSK-986-bound or apo subunit S4-S5 linkers of TRPC3 (light pink) with the activator-bound TRPC6 structure (TRPC6-AM-0883, 6UZ8, cyan) (A) or with the apo TRPC3 structure upon which simulations were based (TRPC3-6CUD, cyan) (B) show slight alterations in conformation. See also Figure S3. (C) The S4-S5 distance is significantly smaller in the GSK-986 simulation subunit occupied by the compound compared to any of the three apo subunits. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test; ****p<0.0001, ***p=0.0003, ***p=0.0008, n=3 independent simulations. Scatter points represent independent replicates and bar graph shows mean ± standard deviation (SD). (D) The GSK-986 simulation subunit occupied by the compound (TRPC3 986 ), subunit A, has a significantly shorter S4-S5 distance than its ligand-free simulation equivalent subunit A. In contrast, the apo subunit S4-S5 distances are not significantly shorter than their ligand-free counterpart subunits. Two-way ANOVA followed by Šídák’s multiple comparisons test, **p=0.0061, n=3 independent simulations. Scatter points represent independent replicates and bar graph shows mean ± SD.

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) TRPC6-AM-1473 (PDB 6UZ8) with SAR7334 analogue AM-1473 (green) in the S1-S3 pocket and the proposed inhibitory lipid (orange) in the S4-S5 pocket. The Q555 TRPC6 equivalent is shown as a potential interacting residue for the lipid. (B) TRPC6-AM-1473 with GSK-986 (purple) from TRPC3 simulations overlaid. Both GSK-986 and the lipid appear to be in contact with Q555. (C) Inhibition of SCA41 by SAR7334 is significantly impaired by mutation Q555A. Two-way ANOVA followed by Šídák’s multiple comparisons test, ***p=0.0005, n=3 independent biological replicates, each being the percentage of cells with nuclear-located GFP-NFAT across 50-200 cells. Scatter points represent biological replicates and bar graph shows mean ± standard deviation (SD). (D) Representative images of NFAT-GFP translocation experiments quantified in (C). Treated cells were fixed and immunostained with antibodies against FLAG and GFP. Nuclei are visualized with DAPI in blue. Image windows are expanded to show NFAT localization in individual cells. Background subtraction was performed equally across all images using ImageJ. Scale bars: 50 μm.

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Residue, Inhibition, Mutagenesis, Standard Deviation, Translocation Assay

Journal: bioRxiv

Article Title: Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

doi: 10.64898/2026.01.07.698177

Figure Lengend Snippet: (A) According to structural comparisons by Bai et. al. and molecular dynamics simulations presented in this study, the closed conformation (dark blue) of TRPC3 is proposed to have the S4-S5 linker in an upward conformation. (B) In the active conformation (light blue), the S4-S5 linker occupies a downward conformation. This is predicted to result in movement of the S5 and S6 helices, analogous to the role of the S4-S5 linker helix in coupling the voltage-sensing domain of the S4 linker to the pore-forming S5 and S6 in voltage-gated channels. The open conformation also likely requires movement of the S6 outward from the pore. (C) GSK-986 interacts with both the S4-S5 linker and the S4 helix. This likely couples the S4-S5 linker to the S4 and maintains the S4-S5 linker in the upward, inhibited conformation. Orange arrows indicate proposed protein-compound interactions. (D) The proposed mechanism of inhibition by GSK-986, and likely other S4-S5 pocket-binding inhibitors of TRPC3 and TRPC6, prevents the downward movement of the S4-S5 linker that is required for activation. Stabilization of the S4-S5 linker may effectively decouple the TRP and S4-S5 linker helices and may also impair outward movement of the S6 helix.

Article Snippet: Additionally, the alignment of TRPC3 GSK-986 with the original cryo-EM structure used for the MD system setup (TRPC3 6CUD ) also showed a slight upward movement of the S4/5 linker, while TRPC3 apo showed higher agreement with the ligand-free, cryo-EM TRPC3 6CUD S4-S5 linker ( ).

Techniques: Inhibition, Binding Assay, Activation Assay