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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

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: The DNA sequence encoding the functional construct of human P2X3 for the structural studies ( ) was synthesized by Genewiz Inc. (Suzhou, China) and subcloned into a pFastBac vector containing a Twin-Strep tag, an EGFP tag, and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus.

Techniques: