New structure enlivens interest in P2X receptors

Abstract

P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use.

Figure 1

Three subunits form one P2X receptor. (a) The trimeric rat P2X2 model, viewed along the axis of threefold symmetry from the extracellular side, with each subunit depicted in a different colour. (b) A single P2X2 subunit, viewed parallel to the membrane plane with the outline dolphin suggested by Kawate et al.. (c) Depiction of the five disulfide bonds within a single P2X2 subunit. Sulfur atoms are shown in grey. (d) Two subunits of the P2X2 receptor presented so as to emphasise the ATP binding pocket which they jointly form. This and subsequent figures show homology models of the rat P2X2 receptor generated with Modeller 9v7 using the zebrafish P2X4.1 crystal structure (PDB accession 3I5D) as a template. MolProbity was used to assess the five lowest-energy models: the selected model showed 98.9% of residues in the allowed regions of the Ramachandran plot. Images were produced using UCSF Chimera 1.4 .

Figure 2

The ATP-binding site. (a) and (b) Space-fill models at 90° to each other viewed parallel to the membrane plane showing the binding pocket for ATP formed between adjacent subunits (shown in blue and yellow). Eight highly conserved residues positioned within the ATP binding pocket are indicated, with the respective ribbon diagrams shown in (c) and (d). We speculate that the negatively charged phosphate groups may interact with the positively charged Lys⁶⁹ and Lys³⁰⁸ residues, whereas Arg²⁹⁰ may interact with the adenine moiety. The ATP molecule (ligand from PDB accession 1B0U) is coloured by element. The amino acid side chains are colour-coded by property as follows: positively charged (green), aromatic (red) and hydrophobic (purple).

Figure 3

The transmembrane domains. (a) TM1 viewed parallel to the membrane with side chains coloured by element. (b) The close proximity of Val⁴⁸ and Ile³²⁸ (purple) of TM1 and TM2 from different subunits, viewed perpendicular to the membrane plane from the extracellular side. (c) TM2 with side chains shown with standard element colours. (d) The narrowest part of the pore with polar gating residues Thr³³⁶, Thr³³⁹ and Ser³⁴⁰ indicated (light blue).

Figure 4

The connecting rods and a possible opening mechanism. (a) A single subunit viewed parallel to the membrane plane, with two connecting rods (the linkers and β-sheets) each rising from the TM1 or TM2 helices. (b) Two connecting rods from two different subunits, which contribute almost all of the eight highly conserved regions within the ATP binding pocket (including the critically implicated Lys⁶⁹ and Lys³⁰⁸ residues). In (a) and (b) the backbone hydrogen bonds are shown (light blue) to emphasise the rigidity within each connecting rod compared with the limited contacts between adjacent connecting rods. Loops and helices that do not form the connecting rods have been omitted for clarity. (c) This highly schematic cartoon shows a possible mechanism for connecting ATP binding to channel gating. ATP binding (lower) repositions Lys³⁰⁸ relative to Lys⁶⁹, and the connecting rod propagates a rotating and separating movement to TM2 relative to TM1.