Journal: bioRxiv

Article Title: Structural basis of lenacapavir-induced HIV-1 capsid disruption during virion maturation

doi: 10.64898/2026.02.16.706074

Figure Lengend Snippet: a–c , Cryo-EM images of the VLPs produced in the absence (a) or presence (b, c) of LEN. Scale bar, 100 nm. d , Cryo-EM density map of the CA lattice within the LEN-containing VLPs, shown from the top (left) and side (right) views. Scale bar, 100 Å. e , The atomic model of a CA hexamer in the LEN-bound lattice fitted into the cryo-EM density map shown in (d). Scale bar, 50 Å. f , Close-up view of the cryo-EM map of a LEN-bound CA lattice (d) and the atomic model (e) showing the density corresponding to a bound LEN. g , Close-up view of the cryo-ET map of a LEN-free CA lattice (EMDB: 13423) and its atomic model, showing the absence of density corresponding to the bound LEN. h , Comparison of two adjacent CA hexamers within the LEN-bound (blue) and LEN-free (yellow; EMDB: 13423) lattices, showing that LEN binding induces a flattening of the curvature between neighbouring hexamers.

Article Snippet: We then determined the capsid lattice structure within VLPs produced in the presence of LEN using a single-particle cryo-EM approach ( , Figs. S2-3, Table S1).

Techniques: Cryo-EM Sample Prep, Produced, Tomography, Comparison, Binding Assay

Journal: bioRxiv

Article Title: Structural basis of lenacapavir-induced HIV-1 capsid disruption during virion maturation

doi: 10.64898/2026.02.16.706074

Figure Lengend Snippet: a , Overview of intra- and interhexamer interfaces within the HIV-1 capsid. b , Comparison of the CTD–CTD dimer interface between CA molecules mediated by helix 9, based on the cryo-EM structure of the LEN-bound CA lattice and the cryo-ET structure of a native CA lattice (EMDB: 13423). The LEN-bound CA lattice (blue) was superimposed onto the native CA lattice (yellow) to visualize structural differences at the dimeric interface. Structures were superimposed by aligning one helix 9 of the CA-CTD of hexamer 1. c , Comparison of the CTD–CTD trimer interface between CA molecules mediated by primary helix 10, based on the cryo-EM structure of the LEN-bound CA lattice and the cryo-ET structure of a native CA lattice (EMDB: 13423). The LEN-bound CA lattice (blue) was superimposed onto the native CA lattice (yellow) to visualize structural differences at the trimeric interface. Structures were superimposed by aligning one helix 10 of the CA-CTD of hexamer 1. d , Comparison of the NTD–NTD interface between CA molecules mediated by helices 2 and 3, based on the cryo-EM structure of the LEN-bound CA lattice and the cryo-ET structure of a native CA lattice (EMDB: 13423). The LEN-bound CA lattice (blue) was superimposed onto the native CA lattice (yellow) to visualize structural differences at the trimeric interface. Structures were superimposed by aligning one helix 3 of the CA-NTD.

Article Snippet: We then determined the capsid lattice structure within VLPs produced in the presence of LEN using a single-particle cryo-EM approach ( , Figs. S2-3, Table S1).

Techniques: Comparison, Cryo-EM Sample Prep, Tomography

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Molecular assemblies and pharmacology of cerebellar GABA A receptors

doi: 10.1073/pnas.2524504123

Figure Lengend Snippet: α1-containing cerebellar GABA A Rs show distinct α and β subunit combinations. Cryo-EM data analysis of the PZ-II-029/GABA dataset identifies five distinct receptor assemblies. ( A ) Two predominant assemblies with well-defined subunit identity, β2-α1-β2-α1-γ2 (viewed from the extracellular space, subunits counted counter-clockwise) and β2-α1-β1-α6-γ2. ( B ) Additional assemblies showing ambiguous density at one or both β subunit positions, corresponding to β2-α1-β2/3-α1-γ2, β1-α1-β1/2-α1-γ2, β1/2-α1-β2/3-α1-γ2. When two subunits are listed at one position, the first one denotes the predominant identity. Percentages of each receptor assembly are calculated based on the number of final particles used for the cryo-EM reconstruction ( SI Appendix , Fig. S5 ). The extracellular domain (ECD) is colored based on the subunit identity. All N -glycosylation is colored in teal, and representative glycosylation at the ECD periphery is labeled with an arrow.

Article Snippet: In data analysis for both datasets ( SI Appendix , Figs. S3–S5 ), more than 2 million GABA A R particles exhibiting salient receptor features in 2D class averages are obtained following a simple cryo-EM cleanup workflow ( SI Appendix , Figs. S3 A and S5 A ).

Techniques: Cryo-EM Sample Prep, Glycoproteomics, Labeling

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet: a , Schematic for purification of the stable-intron complex from saturated cultures. MBP-MS2, Maltose-binding protein fused to MS2 coat protein; ProtA-PP7, 2X ProteinA fused to PP7 coat protein. b , Similar behaviors of tagged stable intron and endogenous stable intron. Shown is an RNA blot of a denaturing gel resolving total RNA from log-phase (LP) and saturated (S) cultures of a wildtype strain that expressed the PP7-GFP-SAC6-MS2 pre-mRNA described in (A). The blot was probed for the MS2 tag (top), and then reprobed for the endogenous SAC6 intron (upper middle), PP7 tag (lower middle), and 5.8S rRNA (bottom), which served as a loading control. c , Proteins in the stable-intron complex. Shown is an SDS-PAGE gel that resolves the cryo-EM sample, stained with Imperial Stain. Bands were labelled based on comparison to the distinct migration patterns of spliceosomal proteins observed in previous spliceosome preparations , . d , e , Enrichment of SAC6-MS2 stable intron ( d ) and snRNAs ( e ) in purified stable-intron complex. Shown is an RNA blot of a denaturing gel resolving the indicated proportion of the glycerol cushion fraction (Input) and the concentrated and dialyzed sample used for cryo-EM analysis (Cryo). The blot was probed using an oligonucleotide that hybridized to a sequence common to the endogenous SAC6 intron and the tagged SAC6-MS2 intron ( d ), and then stripped and reprobed for U1, U2, U4, U5 and U6 snRNAs simultaneously ( e ). Migration of markers with lengths indicated (nucleotides) is at the left. f , No detectable upstream exon in the purified stable-intron complex. Shown is a Urea-PAGE gel resolving the products of primer extension across the GFP-SAC6-MS2 exon–intron boundary at the 5′SS, performed in the absence of dGTP on RNA extracted from equal fractions of the glycerol cushion fraction (input) and IgG affinity flowthrough (exon depleted), and a five-fold fraction of the purified stable-intron complex (cryo-EM sample), as well as on in-vitro-transcribed RNAs representing the intron and the pre-mRNA (standards). Products terminating at the 5′-end of the excised intron (+6) represent the excised intron, and products terminating at the upstream C nucleotides of the 5′ exon (+11 and +20) represent the unspliced pre-mRNA. The +20-extension product was a result of reverse transcriptase readthrough of the first C at the +11 position, as verified by results from the in-vitro-transcribed standards. The in-vitro-transcribed intron standard consisted of the SAC6-MS2 intron with a precise 5′-end defined by hammerhead ribozyme cleavage, and the pre-mRNA standard consisted of an RNA spanning 166 nucleotides upstream to 66 nucleotides downstream of the intron.

Article Snippet: The fractions were analyzed using SDS-PAGE followed by silver staining, and the second and third fractions, which contained the bulk of the purified spliceosomes, were concentrated using a 100 kDa molecular-weight cutoff Ultra Centrifugal Filter (Amicon) and dialyzed against cryo-EM buffer (20 mM HEPES-KOH pH 7.9, 75 mM KCl, 0.25 mM EDTA) for 3 h using a 20-kDa molecular-weight cutoff Slide-a-Lyzer Mini (Thermo).

Techniques: Purification, Binding Assay, Northern blot, Control, SDS Page, Cryo-EM Sample Prep, Staining, Comparison, Migration, Sequencing, In Vitro, Reverse Transcription

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet:

Article Snippet: The fractions were analyzed using SDS-PAGE followed by silver staining, and the second and third fractions, which contained the bulk of the purified spliceosomes, were concentrated using a 100 kDa molecular-weight cutoff Ultra Centrifugal Filter (Amicon) and dialyzed against cryo-EM buffer (20 mM HEPES-KOH pH 7.9, 75 mM KCl, 0.25 mM EDTA) for 3 h using a 20-kDa molecular-weight cutoff Slide-a-Lyzer Mini (Thermo).

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet: a , Resolvability of modelled RNAs and proteins in the stable-intron-complex states I and II and the yeast B act complex. Plotted are the mean Q-score, a measure of resolvability , and fraction of modelled residues for each protein and RNA in the indicated complex. Because the SF3B, SF3A and RES-complex proteins were built using the focus-refined SF3B-lobe cryo-EM density shared by state I and state II , only one value is reported for each protein of this lobe. b , Low-pass filtered cryo-EM density maps of state I and state II of the stable-intron complex and the yeast B act complex, colored by the associated models. Large-scale differences between the density maps are indicated using a black outline. c , Relative position and flexibility of the SF3B lobe of state I and state II of the stable-intron complex. Shown are the atomic models of the well-resolved core and SF3B lobe of the stable-intron complexes, and the unsharpened densities coloured according to the molecular models. The arrow indicates the shift in the position of the SF3b lobe in state II relative to state I. d , Shift of the Prp8 endonuclease domain towards a C-or P-complex-like position in state II of the stable-intron complex. Shown are molecular models of the Prp8 N-terminal domain (N), endonuclease domain, reverse transcriptase domain (RT) and helical bundle domain (HB) in the two states of the stable-intron complex and in the yeast B act , C and P complexes , , . Models were aligned on Prp8. The arrow indicates the shift in the position of the Prp8 endonuclease domain in state II relative to state I. e , Shift in stable-intron complex Prp8 residues 1615–1624 towards the RNA positioned in the 5′-exon-binding site. For clarity, only the nucleobases from state II of the stable-intron complex are shown. f , Weak density for Cwc24 and the 5′ GU of the 5′SS in state II of the stable-intron complex.

Article Snippet: The fractions were analyzed using SDS-PAGE followed by silver staining, and the second and third fractions, which contained the bulk of the purified spliceosomes, were concentrated using a 100 kDa molecular-weight cutoff Ultra Centrifugal Filter (Amicon) and dialyzed against cryo-EM buffer (20 mM HEPES-KOH pH 7.9, 75 mM KCl, 0.25 mM EDTA) for 3 h using a 20-kDa molecular-weight cutoff Slide-a-Lyzer Mini (Thermo).

Techniques: Cryo-EM Sample Prep, Reverse Transcription, Binding Assay

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet: a , Active-site organization for state I of the stable-intron complex. Shown are the overall model for the RNA active site (above) and the cryo-EM density for the active site residues (below). b , Active-site organization for state II of the stable-intron complex; otherwise, as in a. c , Active-site organization of the yeast B act complex in a view analogous to that of a. d , e , Structure of the U6 internal stem loop (ISL) in either the stable-intron complex ( d ) or the B act complex ( e ) in their respective cryo-EM densities. f , Secondary structure of the U6 ISL in the indicated spliceosome structures , – . Dots indicate non-Watson–Crick base pairs.

Article Snippet: The fractions were analyzed using SDS-PAGE followed by silver staining, and the second and third fractions, which contained the bulk of the purified spliceosomes, were concentrated using a 100 kDa molecular-weight cutoff Ultra Centrifugal Filter (Amicon) and dialyzed against cryo-EM buffer (20 mM HEPES-KOH pH 7.9, 75 mM KCl, 0.25 mM EDTA) for 3 h using a 20-kDa molecular-weight cutoff Slide-a-Lyzer Mini (Thermo).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The spliceosome assembles on excised linear introns to protect them from degradation

doi: 10.64898/2026.01.21.700889

Figure Lengend Snippet: a , Sequence determination of RNA bound in the stable-intron complex using the stable-intron complex state II cryo-EM density map. Nucleotides at which the GFP 5′-exon sequence disagrees with the resolved density are labeled in red. b , RNAs observed in preparations of stable-intron complex. The scatter plot shows the abundance of RNAs observed in smaller-scale preparations of the SAC6-MS2 and the ECM33-MS2 stable-intron complexes, as determined by small RNA sequencing (sRNA-seq; expression cutoff, 1 transcript per million (TPM) in both samples). In these preparations, the stable introns were expressed from URA3 exons, and an exon-depletion step was not implemented. CUT314 RNA (red) was abundant in both preparations. c , sRNA-seq reads mapping to the CUT314 locus. A representative downsampled set of 100 mapped reads are shown. The sequence logo was calculated from reads aligning to the 3′-end of the CUT314 locus. d , No detectable effect of CUT314 deletion on stable-intron accumulation. Shown is a blot of a denaturing gel resolving RNA from log-phase (LP) and saturated (S) cultures of the wildtype strain (WT) and a strain in which a genomic region spanning the full-length CUT314 RNA was precisely deleted. The blot was probed simultaneously for the endogenous ECM33 (top) and SAC6 introns (second) and then stripped and reprobed for the CUT314 RNA (third) and 5.8S rRNA (bottom), which served as a loading control. n = 1 biological replicate.

Article Snippet: The fractions were analyzed using SDS-PAGE followed by silver staining, and the second and third fractions, which contained the bulk of the purified spliceosomes, were concentrated using a 100 kDa molecular-weight cutoff Ultra Centrifugal Filter (Amicon) and dialyzed against cryo-EM buffer (20 mM HEPES-KOH pH 7.9, 75 mM KCl, 0.25 mM EDTA) for 3 h using a 20-kDa molecular-weight cutoff Slide-a-Lyzer Mini (Thermo).

Techniques: Sequencing, Cryo-EM Sample Prep, Labeling, RNA Sequencing, Expressing, Control

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