An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

Nagaya, Naomi; Tittle, Rachel K.; Saar, Nir; Dellal, Shlomo S.; Hume, Richard I.

doi:10.1074/jbc.m504545200

Cited by 84 publications

(99 citation statements)

References 33 publications

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“…Unlike what we have seen previously with the double mutant P2X 1 receptors (Marquez-Klaka et al 2007), a strong monomeric band (50.6 § 0.2% of the total P2X protein) of the P2X 2 K69C/F289C subunits remained, indicating that the cross-linking was less eVective than in P2X 1 Rs. A faint additional band corresponding in size to a tetramer has also been observed previously and most likely represents an incompletely cross-linked trimer (Nagaya et al 2005;Marquez-Klaka et al 2007). In support of this interpretation, only complexes corresponding to trimers were detected by BN-PAGE analysis of metabolically labelled P2X 1 complexes (Marquez-Klaka et al 2007) and P2X 2 complexes (not shown).…”

Section: Resultsmentioning

confidence: 74%

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

2008

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P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulWde cross-linking between neighbouring P2X 1 subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X 2 (K69C, F289C), P2X 3 (K63C, F280C), and P2X 4 (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the puriWed receptors revealed a speciWc dimer formation between P2X 2 K69C and P2X 2 F289C mutants. Likewise, co-expression of P2X 1 K68C and P2X 2 F289C, but not P2X 1 F291C and P2X 2 K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X 1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Crosslinking was less eYcient between P2X 3 or P2X 4 mutants but could be enhanced by the short cysteine-reactive crosslinker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X 1 are preserved in P2X 2 receptors and at one of two possible interfaces in heteromeric P2X 1/2 receptors but appears to be redundant for P2X 3 and P2X 4 receptor function.

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Section: Resultsmentioning

confidence: 74%

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

2008

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“…Although we have not determined the coordinating residues responsible for this weak and rapidly reversible potentiation in the wild-type channel, a simple explanation that would be consistent with the present study is that H33 and C348C can form a weak intrasubunit Cd 2+ bridge that stabilizes the open state. Concatamers were constructed as described previously (7,37,38) and were confirmed by restriction digests and DNA sequencing. In addition, cell lysates were evaluated with SDS/PAGE and Western blot analysis, which confirmed that the most abundant species of rP2X2 receptors in HEK cells expressing the concatenated trimeric construct corresponds to the molecular weight of a trimer (Fig.…”

Section: Methodsmentioning

confidence: 99%

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

Heymann

Dai

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergentsolubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATPbound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membraneembedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.pore-opening mechanism | transmembrane intersubunit crevices P 2X receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the immune system, where they play a range of important roles in sensory signaling and inflammation (1, 3).Recent X-ray structures of the zebrafish P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral flexing of the β-sheet connecting the extracellular domain to the TM domain (5). 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 (6-8). 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). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound struc...

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“…Upon ATP binding, structural rearrangements of the subunit interface (3)(4)(5) lead to the opening of the ion channel (6)(7)(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).…”

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confidence: 99%

Agonist trapped in ATP-binding sites of the P2X2 receptor

Jiang

Lemoine

Martz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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...

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An Intersubunit Zinc Binding Site in Rat P2X2 Receptors

Cited by 84 publications

References 33 publications

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

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

Agonist trapped in ATP-binding sites of the P2X2 receptor

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