Molecular mechanism of ATP binding and ion channel activation in P2X receptors

Abstract

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca+2. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

2′,3′-O-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents

“…This study permitted the analysis of the binding mode of the agonist ATP and the antagonist TNP-ATP at the P2X3 receptor. The mechanism of TNP-ATP antagonism, proposed on the basis of its ability to prevent rearrangement of P2X subunits [56], was confirmed by P2X3 modelling studies [57]. The comparison of the binding mode of ATP and TNP-ATP led to an interpretation of the role of the 2′,3′-O-substituent in the antagonist molecule.…”

“…2). In summary, the compound presents a similar binding mode to the one of ATP, observable from X-ray data (zP2X4) and from ATP-bound homology models of human [57] and mouse (Supporting Material) P2X3 receptors developed using the zP2X4 X-ray structure in its active state (PDB code: 4DW1; 2.8-Å resolution [56]) as a template. The phosphate groups of the ligand are involved in a series of polar interactions with the binding cavity residues, most of them being positively charged residues (i.e.…”

“…The 2′-and 3′-hydroxy groups of ATP are missing as the two oxygen atoms are bound to the trinitrophenyl function, which is inserted between two segments of adjacent P2X3 monomers and interacts with the residues located in its proximity. At the same time, this group prevents the possible approach of the two monomers observed during the activation process of the receptor [56]. To verify that the role of the trinitrophenyl group is mainly related to its steric hindrance and not to other features given by its chemical structure (i.e.…”

“…Hence, we designed ATP derivatives bearing in the 2′,3′-O-position bulky groups like a cyclohexylidene or a benzylidene function (2′,3′-Ocyclohexylidene ATP and 2′,3′-O-benzylidene ATP, compounds 4, 9 and 10, respectively, see Schemes 1 and 2). The designed compounds were docked into the binding site of homology models of the human and mouse P2X3, built using the X-ray structure of the zP2X4 receptor in its apo (inactive) state (PDB code: 4DW0; 2.9-Å resolution [56]) as a template. Figure 2 reports a comparison of the simulated binding mode of the TNP-ATP and compound 4 at the human P2X3 binding site.…”

“…In 2009 and 2012, the crystal structures of the zebrafish P2X4 receptor (zP2X4) in the presence and absence of ATP were reported, providing structural details of these membrane proteins [55,56], useful for the analysis of the ATP binding mode and the interpretation of mutagenesis data [12]. Furthermore, a comparative analysis of the open (active state) and closed (inactive state) receptor conformations provides a detailed view of the mechanism of P2X receptor activation.…”