a, b, c, Whole cell recordings of ATP-evoked current (1mM, 3sec, grey bars) from the full-length zfP2X4.1-EGFP construct (a), the ΔzfP2X4-EGFP-A construct (b), and the ΔzfP2X4-EGFP-B construct (c). d, FSEC profiles for zfP2X4.1-EGFP (grey), ΔzfP2X4-EGFP-A (black), and ΔzfP2X4-EGFP-B (blue) expressed in tsA201 cells. The arrows indicate the estimated elution position of the void volume, the zfP2X4-EGFP receptor (trimer) and free EGFP. e, 2Fo-Fc electron density map contoured at 1.2 σ. The blue line represents the Cα trace and the grey bars suggest the boundaries of the outer (out) and inner (in) leaflets of the membrane bilayer. The featured slice depicts TM2 helices but not TM1.

a, Stereoview of the homotrimeric ΔzfP2X4 structure viewed parallel to the membrane. Each subunit is depicted in a different colour. N-acetylglucosamine (NAG) and glycosylated asparagine residues are shown in stick representation. The grey bars suggest the boundaries of the outer (out) and inner (in) leaflets of the membrane bilayer. b, Stereoview of the homotrimeric ΔzfP2X4 structure parallel to the molecular three-fold axis from the extracellular side of the membrane.

a, The ΔzfP2X4 subunit has a dolphin-like shape. Alpha helices (TM1-2 and α2–5), beta strands (β1–14), disulfide bonds (SS1–5), and attached glycans (g2 and 4) are indicated. b, Interface of two adjacent subunits. Subunit A and B are shown in a solvent-accessible surface model and a cartoon representation, respectively. The three major subunit-subunit interfaces are emphasized in different panels where the red ellipsoid highlights the interface between the two subunits. Models are coloured according to domains as in panel (a).

a, A sagittal section reveals a closed conformation of the pore and shows that the gate is located about halfway across the membrane bilayer. Three vestibules (upper, central and extracellular vestibules) are located on the molecular 3-fold axis, with the extracellular vestibule connected to the bulk solution through a fenestration (orange arrow). b, Pore lining surface calculated by the Hole49 program. Each colour represents a different radius range measured from the receptor centre (red: <1.15 Å, green: 1.15–2.3 Å, and purple: >2.3 Å). c, Cartoon representations of the transmembrane domain viewed parallel to the membrane plane. P337 and N341 are shown in grey and potential gate residues (L340, G343, A344, and A347) are shown in green. d, Transmembrane domain viewed perpendicular to the membrane plane.

a, Anomalous difference Fourier map contoured at 8.0 σ (purple) and the modeled Gd³⁺ ions (yellow). b, A peripheral Gd³⁺ binding site in chain B. Distances between Gd³⁺ and the coordinating carboxyl or hydroxyl groups are shown in angstrom. NAG, N-acetylglucosamine c, The central Gd³⁺ binding site is coordinated by three E98 residues. d, View of the Gd³⁺ binding sites in the homotrimeric ΔzfP2X4 structure parallel to the molecular three-fold axis from the extracellular side of the membrane. e, f, Gd³⁺ antagonizes ΔzfP2X4-EGFP whole cell currents measured by patch-clamp electrophysiology. ATP (30µM, 1 sec, black bar) evokes an inward current in tsA201 cells expressing ΔzfP2X4-EGFP (e). Pre-application of Gd³⁺ (100µM, 5 sec, grey bar) inhibits the current evoked by ATP (30µM, 1 sec, black bar) (f). g, Acidic surface on the middle vestibule of ΔzfP2X4. Electrostatic potential surface and cartoon representations of ΔzfP2X4 sliced as in Figure 4 c show an acidic patch located in the middle of the three subunits. The surface is coloured based on the electrostatic potential contoured from −30 kT (red) to +30 kT (blue). White denotes 0 kT. Surface potential was calculated using APBS tools50 for a ΔzfP2X4 model in which side chain atoms were added to residues without side chain atoms in the crystal structure. The following regions were excluded from the calculation: Y53, N78, and N187.

a, A plausible ATP binding pocket located between two neighbouring subunits is highlighted in the black rectangle on an electrostatic potential surface representation of the trimeric ΔzfP2X4-B receptor. The surface is coloured based on the electrostatic potential contoured from −30 kT (red) to +30 kT (blue). White denotes 0 kT. An ATP molecule, scaled appropriately, is also shown. b, A surface representation viewed ~45º from panel (a). The head, dorsal fin and left flipper domains forming the "jaw" shaped ATP-binding pocket are coloured as in Fig. 3. The putative ATP-binding residues are in blue for subunit A (N296, R298, K316) and in red for subunit B (K70, K72, T189). c, Close-up view of the highlighted region in a illustrating subunit A (blue) and B (red). Conserved residues implicated in ATP binding42–45 are labeled, and side chains are in stick representation. Contours from a 2Fo-Fc electron density map drawn around the side chains are in green. The electron density for the side chain of K70 is weak and it has been built as an alanine. d, Conserved residues, shown in space filling representation, are located between the ATP binding site and the transmembrane domain - extracellular domain interface. Only residues for a single subunit are shown.