Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization

Abstract

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X₇ receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X₇ receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

Keywords: Apoptosis; Cryoelectron microscopy; Cytoplasmic domain; Desensitization; Guanosine nucleotides; Ligand gated ion channels; Palmitoylation; Purinergic (P2X) receptors; Structural biology; Zinc ion complex.

Figure 1.. Overall Architecture of rP2X₇ in the Apo State.

(A-D) The three-dimensional reconstruction of rP2X₇ in the apo state viewed parallel to the membrane (A), perpendicular to the membrane from the extracellular surface (B), from the center of the transmembrane domain, toward the cytoplasm (C), and from the intracellular side of the membrane, toward the cytoplasm (D). (E) Ribbon representation of a receptor corresponding to the apo state reconstruction shown in (A). Each protomer is colored differently. (F) A close-up side view of the cytoplasmic portion of the transmembrane domain highlighting how the cytoplasmic cap is composed of domain-swapped β-strands (β₋₁, β₀, and β₁₅) from each protomer. To improve visual clarity of the cytoplasmic cap, the C-cys anchor and cytoplasmic ballast have been removed. (G) Top-down cross-sectional view of the cytoplasmic cap looking toward the cytoplasm. Residues 29–345 and 395–595 of the receptor have been removed to improve visual clarity. Each protomer is a different color. (H) To better visualize the spatial interactions between protomers, one protomer is shown in blue while the other two are shown in gray. (I) Ribbon representation of a single subunit of rP2X₇ receptor, highlighting P2X receptor domains, including two new domains found only in the P2X₇ subtype, the C-cys anchor and the cytoplasmic ballast. The major domains within a protomer are organized by color: extracellular domain in blue, transmembrane domain in green, cytoplasmic cap in cyan, C-cys anchor in purple, and the cytoplasmic ballast in red. The palmitate groups are colored in sand.

Figure 2.. Ion Channel Pore.

(A-F) Cartoon representation of rP2X₇ structures shown parallel to the membrane as a side view, top down view of the ion channel pore, and ion permeation pathway, respectively, are drawn comparing the apo, closed state (A,C,E) to the ATP-bound, open state (B,D,F). Binding of agonist induces conformational changes around the binding pocket that result in outward flexing of the extracellular domain’s core β-strands, pulling on TM2, causing outward helix expansion to open the pore. For the pore size plots, colors represent different radii: reddish pink < 1.15 Å, green between 1.15 – 2.30 Å, and purple > 2.30 Å. The N-terminus, TM1, and cytoplasmic ballast have been removed from panels (E) and (F) to improve visual clarity.

Figure 3.. Extracellular Orthosteric Ligand-Binding Site.

(A-B) View of the orthosteric binding pocket comparing key interactions to ATP in rP2X₇ (A) and hP2X₃ (B). Two conserved phenylalanine residues (F188 and F293; rP2X₇ numbering), proposed to stabilize the ATP-binding pocket, are also similar in both structures (residues not shown). (C-F) Surface representation of the binding pocket for rP2X₇ in the open state (C) and apo state (E) compared to hP2X₃ in the open state (D) and apo state (F). The apo binding pocket of rP2X₇ contains a narrow channel (< 11 Å orifice) which shields the pocket from solvent, limiting ligand access. The apo binding pocket of hP2X₃ is significantly more solvent exposed (17 Å orifice). The orthosteric-binding pocket is at a subunit interface, with protomer A in green for open state of rP2X₇ and forest green for open state of hP2X₃ or red for the apo state of rP2X₇ and red-purple for the apo state of hP2X₃. Protomer B is in gray and protomer C is in white.

Figure 4.. P2X₇ Cytoplasmic Domain – the C-cys Anchor is Palmitoylated to Modulate Receptor Function by Preventing Desensitization.

(A) Surface representation of the apo state of rP2X₇ highlighting locations of the C-cys anchor and putative phospholipid binding site. (B, left panel) The C-cys anchor is a loop containing six cysteine residues and one serine residue that each face toward the plasma membrane. (B, right panel) At least four cysteines (C362, C363, C374, and C377) and one serine (S360) on the C-cys anchor are palmitoylated. Palmitoylation of the C-cys anchor is also present in the open state reconstruction but more easily visualized in the higher resolution apo state map. The higher resolution apo state reconstruction also reveals that at least one cysteine (C4) in the N-terminus is palmitoylated. Because of domain swapping within the context of a trimeric receptor, the N-terminus of one protomer is adjacent to the C-cys anchor of an adjacent protomer, grouping together the palmitoylated moieties of different protomers and effectively interlocking them to the membrane. (C-D) Deletion (rP2X₇-ΔCcys) of the C-cys anchor (C) or mutation of residues in the C-cys anchor to alanine (rP2X₇-CcysMut) to prevent their palmitoylation (D), both result in channels that, when expressed in Xenopus oocytes, nearly completely desensitize during a 30 second application of 100 μM ATP (black bar). Wild type rP2X₇ receptor (rP2X₇-WT) does not desensitize. (E) The putative phospholipid binding site, modeled as a PS, is near the middle of the plasma membrane. Each protomer is a different color.

Figure 5.. P2X₇ Cytoplasmic Domain – the Cytoplasmic Ballast Contains a Novel Fold.

(A) Ribbon representation of one subunit of the open state structure of rP2X₇ receptor shown in orthogonal views. Each of the major P2X₇ domains is color-coded. The cytoplasmic cap, C-cys anchor, and cytoplasmic ballast domains are highlighted. (B) Ribbon representation of the cytoplasmic ballast of rP2X₇ receptor displaying the secondary structure elements, the two zinc ions, and the guanosine nucleotide. (C) Topology diagram of the cytoplasmic ballast, shown in the same orientation as (B), reveals a novel fold without structural homology in the PDB. Dashed lines denote un-modeled regions. (D-E) Additional ribbon representations of the cytoplasmic ballast of the rP2X₇ receptor, shown in two different orthogonal views from (B), highlighting the relative locations of the zinc ions and the guanosine nucleotide within the ballast fold. The models are missing residues S443 to R469 in the apo state and residues S443 to R471 in the open state.

Figure 6.. The P2X₇ Cytoplasmic Ballast Contains a Dinuclear Zinc Ion Complex and a High-Affinity Guanosine Nucleotide Binding Site.

(A) The cytoplasmic ballast from each protomer lies beneath the TM domain from an adjacent protomer. (B) The entire cytoplasmic domain viewed perpendicular to the membrane from the cytoplasm. Residues S443 - R469 are missing from the apo state structure, visualized here, because no density is present in the map. (C) A bottom up view focused on the center of the cytoplasmic domain, perpendicular to the membrane from the cytoplasm reveals how, in the context of a trimer, helices α₁₂ and α₁₃ of each cytoplasmic ballast form a hexagonal hole (diameter ~ 14 Å) through which the α₉ helix from each protomer (labeled A, B, and C) emerges. This is referred to as the “cytoplasmic plug”. The entire cytoplasmic domain assembles as a trimer through, in part, the interactions at the interface formed by helices α₉, α_12, and α₁₃. (D) The cytoplasmic dinuclear Zn²⁺ ion complex is coordinated by seven cysteine residues in a tetrahedral geometry. (E-F) The cytoplasmic guanosine nucleotide binding site, occupied by a GDP molecule, shown in two views highlighting the density for GDP (E) and key interactions made with conserved P2X₇ residues (F). (G) Measurement of ³H-GDP saturation binding to purified, detergent solubilized rP2X₇ receptor using SPA. The K_d for GDP binding was directly measured as 40 ± 10 nM. Error bars represent the standard deviation of three measurements. (H) Inhibition of ³H-GDP binding to rP2X₇ receptor by guanosine nucleotides yields K_i values of 70 ± 10 nM and 150 ± 15 nM for GDP and GTP, respectively. Non-guanosine diphosphate nucleotides do not displace ³H-GDP and thus do not bind to the cytoplasmic domain of P2X₇ receptor. Error bars represent the standard deviation of three measurements. (I) The guanosine nucleotide binding pocket is exposed at the interface between two cytoplasmic ballasts, accessible as a docking site for protein-protein interactions. Each protomer is a different color.

Figure 7.. Molecular Mechanism of P2X Receptor Gating.

(A-B) Cartoon models summarizing the gating mechanism for the desensitizing P2X₁₋₆ receptor subtypes (A) compared to the non-desensitizing P2X₇ receptor subtype (B).