The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

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...

Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as -methyl-d-glucamine (NMDG). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG permeability superimposes that of Na and demonstrate that the molecular motions leading to the permeation of NMDG are very similar to those that drive Na flow. We found, however, that NMDG "percolates" 10 times slower than Na in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu 63 and Arg 274 produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu 63 and Arg 274 contributes to the ion channel function. Engineered cysteines E63C and R274C formed redoxdependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu 63 and Arg 274 , allowing the transmembrane helices to open the channel.P2X receptors (P2XRs) 5 are membrane cation channels gated by extracellular ATP. They are widely distributed in excitable and nonexcitable cells of vertebrates (1) and play key roles in synaptic transmission (2), presynaptic modulation (3), taste sensation (4, 5), pain signaling (6, 7), and intestinal motility (8).P2XRs are allosteric trimeric ion channels formed by the oligomerization of three identical or homologous subunits (9, 10). Each subunit (there are seven identified so far in mammals, termed P2X1 through P2X7) possesses intracellular N and C termini and two transmembrane segments, termed TM1 and TM2, joined by an extracellular ectodomain. The binding of ATP to the ectodomain promotes the rapid opening of the ion channel, referred to as gating. Once the channel is opened, cations transit through the pore down their electrochemical gradients, leading to the transient influx of sodium and calcium into the cell. This in turn leads to depolarization of the cell and downstream calcium signaling. It is thought that gating involves long range conformational changes that are transduced from the ATP-binding sites to the ion channel and even to the cytosolic domain (11). However, the molecular mechanisms underlying the gating process in P2XR are still largely unknown.Very recently, the crystal structure of zebra fish P2X4R (zfP2X4R) has been solved by x-ray crystallography at a resolution of 3.1 Å (12). The structure was solved in the absence of ATP and probably represents the closed state of the ion channel. The location of the ATP-binding sites remains unknown; however, it has been suggested that the nucleotide binds to deep intersubunit grooves, located on the outside of the trimer, 45 Å from the ion channel domain, and surrounded by conserved residues previously shown to be important for ATP function (12). This structure thus represents an outstanding advance...

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.azobenzene photoswitch | purinergic receptors O ptogenetic approaches in neuroscience rely on the heterologous expression of engineered light-gated ion channels or pumps to trigger or inhibit electrical activity of selected neurons (1, 2). This powerful and revolutionizing technique provides an exquisite remote control of neuronal circuits that drive behavior in animals. Of special interest is the optogenetic pharmacology (also known as optochemical genetic) (3, 4), which allows the control of an ion channel or receptor function by a photoswitchable ligand that is irreversibly tethered to the genetically modified protein through cysteine substitution (3, 4). Ligands are pharmacologically active substances targeting either competitive (5-7), or noncompetitive binding sites (8-10), and light sensitivity is mostly conferred by substituting a photoisomerizable azobenzene derivative, which interconverts reversibly between a long trans-isomer and a short cis-isomer (11). However, this approach is technically demanding, as it requires the design of site-specific ligands for each target. The development of versatile methods with generic photoswitchable molecules would thus improve the optochemical strategy.Here, we present optogating, a unique method for the optical control of ATP-activated P2X channel gating. P2X receptors are a family of trimeric, cation-selective channels (12, 13) comprising seven mammalian subtypes that mediate a variety of physiological responses, including fast synaptic transmission, contraction of smooth muscle, modulation of neurotransmitter release, and pain sensation (12,14,15). P2X receptors are considered as emerging therapeutic targets because of their link to cancer (16), inflammatory (17), and neuronal diseases, including neuropathic pain (18). Inspired by previous works showing that chemical modification of the transmembrane (TM) pore region of the P2X2 receptor affects channel gating through labeling of single cysteine mutants by p...

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.DOI: http://dx.doi.org/10.7554/eLife.11050.001

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

The molecular mechanism underlying channel opening in response to agonist binding remains a challenging issue in neuroscience. In this regard, many efforts have been recently undertaken in ATP-gated P2X receptors. Among those efforts, we have provided evidence in the P2X2 receptor that tightening of ATP sites upon agonist binding induces opening of the ion channel. Here we extend our analysis to show that the sulfhydryl-reactive ATP analog 8-thiocyano-ATP (NCS-ATP), a potent P2X2 agonist, when covalently labeled in the ATP-binding site at position Leu186 likely favors the tightening mechanism, but not the channel opening mechanism. Our data predict the existence of intermediate or preactivation state(s) trapped by NCS-ATP, in which tightening of the binding site is favored while the channel is still closed. We propose that this (these) intermediate ATP-bound state(s) prime(s) channel gating in the P2X2 receptor.