Agonist trapped in ATP-binding sites of the P2X2 receptor

Abstract

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATPderived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATPbinding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.affinity labeling | chemical modification | purinergic receptor P 2X receptors are oligomeric ATP-gated ion channels selective to cations (1) and are involved in physiological processes as diverse as synaptic transmission, the response to inflammation, and pain perception (2). Upon ATP binding, structural rearrangements of the subunit interface (3-5) lead to the opening of the ion channel (6-8), but the entire molecular sequence of events that couple ATP binding to channel opening remains unknown. The recent X-ray structure of the P2X4 receptor in a closed resting state represents in this regard a decisive step (9). It confirms the trimeric stoichiometry of the ion channel, in agreement with the fact that there are three activatable ATP-binding sites (10), and provides a structural context to interpret functional data (11).Early studies based on mutagenesis data have identified highly conserved extracellular residues important for ATP function and have proposed that ATP binding occurs through the extracellular domain (12-19), presumably at the subunit interface (15,17). When mapped on the crystal structure, most of these residues are observed to line a large and deep intersubunit cavity shaped like an open "jaw" and located approximately 45 Å away from the ion channel domain (9). This observation thus suggests that these residues participate in ATP binding; however, the crystal structure was solved in the absence of ATP, and therefore no direct evidence support this hypothesis to date.We used the proximity-dependent "tethering" approach (20) to localiz...

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

“…For example, ATP binding to the cleft and subsequent cleft closure were predicted to occur based on proximity tethering (11), normal mode analysis (12), and both metal bridging and spectroscopic studies (13,14). In addition, the accessibility of both methanethiolsulfonate compounds and metals (Ag + and Cd 2+ ) to cysteine residues introduced into TM1 and TM2 (6)(7)(8) is consistent with the expansion of the external pore predicted from the zfP2X4 structures (Fig.…”

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

“…3 B and C) revealed at least two poses that should allow the reaction with the side chain for all models except for the solvent-inaccessible E119C mutant and the A118C mutant, in which only one pose was identified. To further interpret our data, we measured the distance between the maleimide moiety of the docked TMRM and residue F188 (homologous to residue L186 in the P2X2R) as a marker for the ATP binding site (11). This measurement takes into account the flexibility of TMRM that should explore a cone in space after being fixed to the receptor and thus, gives a rough idea of possible interactions of TMRM with the ligands.…”

“…Three of these disulfide bridges are arranged in the head domain (2). Two findings suggest a close association of this region with ATP binding: (i) the P2X7 subtype has been found to be constitutively active on ADP ribosylation of residue R125 located between the first and second conserved cysteine residues (10), and (ii) a recent report has shown that the thiol-reactive ATP analog 8-thiocyano-ATP (NCS-ATP) can be covalently attached to the cysteine-substituted residue N140C in the head domain of the P2X2R (11). To probe the proximity of the first intercysteine region to the presumed ATP binding site and a possible function of the cys-rich region in receptor activation and/or desensitization, we performed VCF with the fast desensitizing P2X1R expressed in Xenopus oocytes.…”

P2X receptors

“…For example, disulfide locking of pairs of engineered cysteine residues has provided direct evidence for the approximation of amino acid residues [32,[77][78][79][80][81][82][83][84]. The openclosed transition of the receptor can also be driven by light in the complete absence of ATP, when a light-sensitive azobenzene molecule is incorporated into the receptor by attachment to cysteine residues in two different subunits [85,86].…”