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...

Background: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H + electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds α-keto acids in vitro.

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...