Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A, B) Overall structure (A) and cryo-EM density map (B) of the sivopixant- and ATP-bound human P2X3 receptor viewed parallel to the membrane. Each subunit is colored distinctly. In A, the sivopixant and ATP molecules are shown in sphere representation. In B, the ligand densities are shown in gray. (C) The dolphin-shaped P2X3 subunit is colored differently according to each structural feature. The cryo-EM densities for sivopixant and ATP are shown. (D) Structure of the sivopixant- and ATP-bound human P2X3 receptor viewed perpendicular to the membrane from the extracellular side. (E) The transmembrane domain structures of the sivopixant- and ATP-bound P2X3 structure (left panel, this study) and the apo P2X3 structure (right panel, PDB ID: 5SVJ) viewed from the extracellular side. (F, G) The overall structure (F) and cryo-EM density map (G) of the ATP-bound human P2X3 receptor viewed parallel to the membrane. Each subunit is colored distinctly. In F, ATP molecules are shown in sphere representation, and the cryo-EM density for ATP is also shown. In G, ATP densities are shown in gray. (H) The transmembrane domain structures of the ATP-bound P2X3 structure (this study, left panel), the desensitized P2X3 structure (middle panel, PDB ID: 5SVL), and the open P2X3 structure (right panel, PDB ID: 5SVK) viewed from the extracellular side.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Cryo-EM Sample Prep, Membrane

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A) Overall structure of the sivopixant- and ATP-bound human P2X3 receptor (upper-left panel) and a close-up view of the sivopixant binding site (lower-left panel) viewed perpendicular to the membrane from the extracellular side. Close-up views of the sivopixant binding site are shown from two different angles (upper-right and lower-right panels). The ligand molecules and amino acid residues involved in sivopixant binding are shown in stick representation. Dotted lines represent hydrogen bonds. (B) Schematic diagram of the interactions between P2X3 and sivopixant. Dotted lines represent hydrogen bonds. (C) Amino acid sequence alignment of P2X3 receptors from Mus musculus (Q3UR32.1), Rattus norvegicus (P49654.1), Canis familiaris (XP_038280235.1), Bos taurus (XP_059731161.1), Gallus gallus (NP_001384137.1), and Danio rerio (NP_571698.3); P2X receptors from Homo sapiens (P2X1: P51575.1; P2X2: Q9UBL9.1; P2X3: P56373.2; P2X4: Q99571.2; P2X5: Q93086.4; P2X6: O15547.2; and P2X7: Q99572.4) as well as the Mus musculus P2X2 receptor (Q8K3P1.2) and Rattus norvegicus P2X2 receptor (CAA71046.1). The residues involved in sivopixant binding are shown. The blue circles indicate the residues shown in B.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Binding Assay, Membrane, Sequencing

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A) Representative current traces of the effects of sivopixant on human P2X3 currents at different ATP concentrations. (B) Effects of sivopixant on ATP (0.1, 1 and 10 µM)-evoked currents of human P2X3 (mean ± SEM, n = 3-4). (C, E) Representative current traces of sivopixant effects at 1 μM (C) and 0.3 μM (E) on ATP-evoked currents of human P2X3 and its mutants (C: M96W, M165W, and Y285W; E: T82I). (D, F) Effects of 1 μM (D) and 0.3 μM (F) sivopixant on the ATP-evoked currents of human P2X3 and its mutants (mean ± SEM, n = 3-5). Two-way ANOVA followed by Tukey‘s multiple comparisons test (B) and one-side one-way ANOVA followed by post hoc test (D, F), **p < 0.01, ****p < 0.0001 vs. WT). The hP2X3 WT data shown in and were obtained from the same cells and are shown in the respective panels for comparison.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Comparison

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A-F) Close-up views of the sivopixant binding site of the sivopixant- and ATP-bound P2X3 structure (in this study, yellow and blue). The human P2X1 structure (PDB ID: 9C2A) (A), the human P2X2 structure (PDB ID: 9DDV) (B), the human P2X4 structure (PDB ID: 9BQH) (C), the human P2X7 structure (PDB ID: 9E3M) (D), and the predicted heterotrimer structure formed by two P2X2 subunits and one P2X3 subunit (AlphaFold3, ipTM=0.71) (E, F) are superposed onto the P2X3 structure and shown in gray. In E, the gray chain superposed onto the yellow chain is the P2X2 subunit, while the gray chain superposed onto the blue chain is the P2X3 subunit. In F, the gray chain superposed onto the yellow chain is the P2X3 subunit, while the gray chain superposed onto the blue chain is the P2X2 subunit.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A) Representative current traces of the effects of sivopixant on ATP-evoked currents of human P2X3 wild type (WT) and its gain-of-function mutant (GOF). (B) Effects of sivopixant on ATP-evoked currents of human P2X3 and its mutant (mean ± SEM, n = 3-4). (One-way ANOVA followed by Tukey‘s multiple comparisons test, ***p < 0.001 hP2X1GOF vs. hP2X1-WT, ****p < 0.0001 hP2X2GOF vs. hP2X2-WT). The hP2X3 WT data shown in and were obtained from the same cells and are shown in the respective panels for comparison.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A) Superimposition of the sivopixant- and ATP-bound P2X3 structure (in this study, blue) and the ATP-bound, open P2X3 structure (PDB ID: 5SVK, red) onto the apo, closed P2X3 structure (PDB ID: 5SVJ, gray) viewed parallel to the membrane. Close-up views of the dorsal fin, left flipper, and lower body domains are also shown. The arrows indicate conformational changes in the open P2X3 structure (red) and the sivopixant- and ATP-bound P2X3 structure (blue). (B, C) Close-up view of the ATP binding site of the sivopixant- and ATP-bound P2X3 structure (in this study, blue) (B) and the open P2X3 structure (PDB ID: 5SVK, red) (C). The ATP molecules and amino acid residues involved in ATP binding are shown in stick representation. Dotted lines represent hydrogen bonds. (D, E) Superimposition of the open P2X3 structure (PDB ID: 5SVK, red) (D) and the sivopixant- and ATP-bound P2X3 structure (this study, blue) (E) onto the apo P2X3 structure (PDB ID: 5SVJ, gray). Only the transmembrane and body domains from the two subunits in the foreground are shown.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Membrane, Binding Assay

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: (A) Close-up view of the sivopixant binding site of the P2X3 receptor. Superimposition of the sivopixant- and ATP-bound P2X3 structure (this study, blue) and the open P2X3 structure (PDB ID: 5SVK, red) onto the apo P2X3 structure (PDB ID: 5SVJ, gray) viewed perpendicular to the membrane from the extracellular side. Dotted lines indicate the distance (Å) between the Cα atoms of Glu289 in two adjacent subunits. (B-E) MD simulations using the sivopixant- and ATP-bound structure with both retained (B), ATP deleted (C), sivopixant deleted (D) and both deleted (E) as starting models. The distance plots of Cα atoms between Glu289 of two adjacent subunits are shown. The average distances in the trimer are shown.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques: Binding Assay, Membrane

Journal: bioRxiv

Article Title: Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

doi: 10.64898/2026.01.03.697462

Figure Lengend Snippet: Cartoon diagrams illustrating the conformational changes of P2X3 from the apo state (middle) to the ATP-bound, open state (left) and the sivopixant- and ATP-bound, closed state (right). The arrows indicate conformational changes between two states.

Article Snippet: For grid preparation, the P2X3 protein was mixed with 0.1 mM Sivopixant (MedChemExpress, China).

Techniques:

Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: Sorted PSN-derived exosomal miR-1306-3p activated spinal P2X3R and enhanced synaptic transmission, thus leading to visceral pain. (A) Schematic diagram and representative images of sorting PSN-derived exosomes from spinal dorsal horn. (B) The expressions of miR-1306-3p, miR-1949, miR-185-5p, and miR-324-5p were detected in the PSN-derived exosomes from spinal dorsal horn of CON and NMD mice by Q-PCR (n = 4 mice for each group, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (C) Fluorescent representation of co-localization of GFP (green), P2X3R (red), and DAPI (blue) in the spinal dorsal horn. Scale bar = 50 μm. Representative 3D confocal images on the right was the enlarged view in the white box of merge. Scale bar = 20 μm. (D) Representative traces of sEPSCs were recorded in the T13 to L2 spinal dorsal horn neurons before and after the incubation of miR-1306-3p and Gefapixant. Histogram of the amplitude and frequency of sEPSCs in Pre, miR-1306-3p, and miR-1306-3p+Gefapixant group (n = 10 cells from 4 mice for each group, ** P < 0.01, *** P < 0.001, one-way ANOVA followed by Tukey post hoc test). (E) Statistical diagram of the visceral pain threshold in NMD mice after injection of antagomir-miR-1306-3p or antagomir-negative control (antagomir-NC) at Pre, 0.5, 1, 2, and 4 hours (antagomir-NC: n = 6 mice, antagomir-miR-1306-3p: n = 7 mice, ** P < 0.01, 2-way ANOVA followed by Sidak post hoc test). (F) Statistical diagram of the visceral pain threshold in NMD mice after injection of Gefapixant at Pre, 0.5, 1, 2, 4, and 8 hours (n = 7 mice for each group, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way ANOVA followed by Dunnett post hoc test). (G) Histogram of the visceral pain thresholds in CON mice treated with agomir-negative control (agomir-NC) or agomir-miR-1306-3p at Pre, 0.5, 1, 2, and 4 hours, respectively (n = 6 mice for each group, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (H) Histogram of the visceral pain thresholds in CON mice treated with agomir-miR-1306-3p+NS or agomir-miR-1306-3p+Gefapixant at Pre, 0.5, 1, 2, and 4 hours, respectively (n = 6 mice for each group, * P < 0.05, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; NMD, neonatal maternal deprivation; PSN, primary sensory neuron; Q-PCR, real-time quantitative polymerase chain reaction; sEPSC, spontaneous excitatory postsynaptic current.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Derivative Assay, Transmission Assay, Incubation, Injection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: siR-Rab27a reduced visible PSN-derived exosomes, suppressed spinal synaptic transmission, and alleviated visceral pain. (A and B) Representative images and fluorescence area of GFP were quantified in the left spinal dorsal horn of the NMD mice injected with siR-NC or siR-Rab27a (siR-NC: n = 3 mice, siR-Rab27a: n = 4 mice, * P < 0.05, 2-sample t test). Scale bar = 50 μm. (C) The protein expression of GFP in the left spinal dorsal horn of siR-NC and siR-Rab27a mice (siR-NC: n = 5 mice, siR-Rab27a: n = 4 mice, * P < 0.05, 2 sample t test). (D) Representative images of GFP, NeuN-labeled neurons, and DAPI-labeled nucleus in the left spinal dorsal horn of siR-NC and siR-Rab27a mice. Scale bar = 50 μm. The white box in the lower right corner was the typical GFP + positive neurons, scale bar = 10 μm. (E) Quantification of the percentage of GFP + positive neurons in the spinal dorsal horn neurons of siR-NC and siR-Rab27a mice (siR-NC: n = 4 mice, siR-Rab27a: n = 5 mice, * P < 0.05, Mann–Whitney test). (F) Representative images of GFP, P2X3R-positive neurons and DAPI in the left spinal dorsal horn of siR-NC and siR-Rab27a mice. Scale bar = 50 μm. The white box in the lower right corner was the typical GFP + P2X3R + positive neurons. Scale bar = 10 μm. (G) Quantification of the percentage of GFP + P2X3R + positive neurons in spinal P2X3R-positive neurons of siR-NC and siR-Rab27a mice (n = 4 mice for each group, * P < 0.05, Mann–Whitney test). (H) Representative traces of sEPSCs were recorded in the T13 to L2 spinal dorsal horn neurons of siR-NC and siR-Rab27a mice. Histogram and cumulative probability distributions of the amplitude and frequency of sEPSCs in siR-NC and siR-Rab27a mice (siR-NC: n = 8 cells from 3 mice, siR-Rab27a: n = 6 cells from 3 mice, * P < 0.05, 2-sample t test). (I) The visceral pain threshold at Pre, 1, 2, 3, 4, and 5 days in NMD mice injected with siR-NC or siR-Rab27a, respectively (n = 8 mice for each group, ** P < 0.01, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (J) Statistical diagram of the time on the rod in the siR-NC or siR-Rab27a group at Pre, 1, 2, 3, 4, and 5 days, respectively (n = 8 mice for each group, P > 0.05, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; GFP, green fluorescent protein; NMD, neonatal maternal deprivation; PSN, primary sensory neuron; sEPSC, spontaneous excitatory postsynaptic current.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Derivative Assay, Transmission Assay, Fluorescence, Injection, Expressing, Labeling, MANN-WHITNEY

Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: A working model showing that exosomes in the circuit of DRG-spinal cord leading to chronic visceral pain by exosome visualization technologies. The upregulation of Rab27a in colon-related DRGs promoted the release of exosomal miR-1306-3p, which activated P2X3R in the spinal dorsal horn neurons and enhanced synaptic transmission, thereby contributing to chronic visceral pain. Meanwhile, intrathecal injection of GW4869 or siR-Rab27a reduced visible PSN-derived exosomes in spinal cord, suppressed spinal synaptic transmission, and alleviated visceral pain in NMD mice. Inhibition of P2X3R also alleviated visceral pain by intrathecal injection of Gefapixant. DRG, dorsal root ganglia; EPSC, excitatory postsynaptic currents; MVB, multivesicular bodies; NMD, neonatal maternal deprivation; PSN, primary sensory neuron.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Transmission Assay, Injection, Derivative Assay, Inhibition