Gating the pore of P2X receptor channels

Abstract

Three families of ligand-activated ion channels mediate synaptic communication between excitable cells in mammals. For pentameric channels related to nicotinic acetylcholine receptors and tetrameric channels like glutamate receptors, the pore-forming and gate regions have been studied extensively. In contrast, little is known about the structure of trimeric P2X receptor channels, a family of channels that are activated by ATP and serve crucial roles in neuronal signaling, pain transmission and inflammation. To identify the pore-forming and gate regions within P2X receptor channels, we introduced cysteine residues throughout the two transmembrane (TM) segments and studied their accessibility to thiol reactive compounds and ions. Our results show that the TM2 helix lines the central ion conduction pore, that the TM1 helix is positioned peripheral to TM2, and that the flow of ions is minimized in the closed state by a gate formed by the external region of TM2.In mammals there are seven subtypes of P2X receptor channels (termed P2X 1-7 ) that are widely expressed throughout the nervous system and many other tissues, including muscle and epithelia 1, 2 . From a structural perspective P2X receptor channels are intriguing because they are formed by three identical or related subunits 3-7 , each having a large extracellular segment of ∼ 280 amino acids that forms the ATP binding domain 8 , with two flanking transmembrane helices (TM1 and TM2) 9, 10 spanning the membrane and leaving the N and C termini on the intracellular side (Fig. 1). Our objective was to determine the relative contribution of the TMs to forming the ion conduction pore in P2X receptor channels and to localize the gate region, two fundamentally important questions that remain unresolved 1 . In the present study we set out to answer these questions by introducing cysteine (Cys) residues throughout both TMs of the P2X 2 receptor channel, an isoform that desensitizes slowly after activation by ATP 2 , and measuring the apparent rate of chemical modification with thiol reactive compounds and ions applied to the external side of the membrane. For this approach to be informative for our purposes, the thiol reactive compounds and ions should 1) not modify the channel unless a Cys is introduced, 2) modify with rates approaching those observed in aqueous solution when the channel is open, and 3) modify an introduced Cys below the gate with dramatically slower rates when the channel is closed 11-13 . These requirements were not achieved in previous Cys accessibility studies on P2X receptors 14-17 , leaving the pore-forming and gate regions unclear 1 . Although modification of residues in both TM1 and TM2 was observed, no rapid modification rates were obtained, leaving open the possibility that reagents access introduced Cys residues through non-aqueous pathways (e.g. protein or lipid) and/or react with rare conformations of the channel. Furthermore, contradictory conclusions were put forward as to the secondary structure of TM2 and the location of the ga...

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

“…For both constructs, application of external Cd 2+ was without effect when applied in the absence of ATP (Fig. 3 A and C), as expected from both structural (4,5) and functional (6)(7)(8) evidence that the gate in P2X receptors is positioned external to this engineered bridging site, and thus closure of the gate in the absence of ATP would prevent Cd 2+ from reaching the bridging site. However, when Cd 2+ was applied externally in the presence of ATP, the metal produced robust potentiation of ATP-activated currents ( Fig.…”

“…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. S1A).…”

“…When expressed in HEK cells, both constructs gave rise to ATP-activated macroscopic currents and exhibited concentration-response relations comparable to those in the wild-type rP2X2 receptor ( Fig. 3 and Table S1) (6). For both constructs, application of external Cd 2+ was without effect when applied in the absence of ATP (Fig.…”

“…The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4,5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6)(7)(8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9,10).…”

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…The second membranespanning domain of the P2X receptor lines the central ion-conduction pore and the gate is positioned deep in the membrane-spanning domain segment, corresponding to the region flanking the SNP [84,85]. Mutations in the region also alter the permeability of P2X receptor channels for divalent ions [86].…”