Thr339-to-Serine Substitution in Rat P2X2Receptor Second Transmembrane Domain Causes Constitutive Opening and Indicates a Gating Role for Lys308

Cao, Lishuang; Young, Mark T.; Broomhead, Helen; Fountain, Samuel J.; North, Richard

doi:10.1523/jneurosci.4036-07.2007

Cited by 51 publications

(75 citation statements)

References 39 publications

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“…These results have partly been conWrmed at P2X 3 , P2X 4 , and P2X 7 receptors (Wilkinson et al 2006;Zemkova et al 2007;Worthington et al 2002). A recent single channel analysis on the background of a spontaneously opening P2X 2 mutant provides good evidence that at least K68 contributes directly to ATP binding while residues corresponding to K309 are more likely to have a function in channel gating (Cao et al 2007). In addition, a conserved arginine residue (R292) and conserved aromatic amino acid residues (F185, F291), in particular, the conserved NFR motif (N290-R292 in P2X 1 ) has been supposed to contribute to ATP binding, possibly by interacting with the adenine ring Evans 2004, 2007).…”

Section: Introductionmentioning

confidence: 92%

“…Alanine mutations of this residue in P2X 1 , P2X 2 , P2X 3 and P2X 4 subunits resulted in functionally impaired or non-responsive receptors (Wilkinson et al 2006;Ennion et al 2000;Jiang et al 2000;Zemkova et al 2007). Most convincing support for a ligand binding function of this residue has been demonstrated by single channel analysis of the K69A mutation in the background of a constitutively active P2X 2 T339S mutant (Cao et al 2007). …”

Section: Cross-linking Eyciencymentioning

confidence: 99%

“…In agreement with our hypothesis that K68 and F291 lie on opposite sites of an intersubunit binding site, a contribution of K68 and K309 from neighbouring subunits to ATP function has also been proposed (Wilkinson et al 2006;Marquez-Klaka et al 2007). However, evidence for a function of residues analogous to P2X 1 K309 and a nearby segment involving G316 to I333 (in P2X 4 ) in channel gating rather than ligand binding is also accumulating (Cao et al 2007;Yan et al 2006). …”

Section: Cross-linking Eyciencymentioning

confidence: 99%

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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: Introductionmentioning

confidence: 92%

Section: Cross-linking Eyciencymentioning

confidence: 99%

Section: Cross-linking Eyciencymentioning

confidence: 99%

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

2008

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“…It is also well established that the P2X pore is lined by TM2 (19 -22), with TM1 making little contribution to ion flow (23,24). Moreover, careful studies of permeation and selectivity have suggested that the same area of TM2 is responsible for both ion selection and channel opening (25)(26)(27)(28)(29)(30)(31). Mutational approaches have shed light on the extracellular domain of P2X receptors, including how the ATP binding pocket may form (32), how the 10 conserved cysteines contribute to the fold of the protein (33,34), and how cations such as Zn 2ϩ profoundly affect receptor function (35)(36)(37)(38).…”

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confidence: 99%

Gated Access to the Pore of a P2X Receptor

Kracun

Chaptal

Abramson

et al. 2010

Journal of Biological Chemistry

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P2X receptors (P2X1-P2X7) are cell surface cation channels that transition from closed to open states upon binding ATP. They represent a physiologically and medically important (1), as well as a structurally distinct family of ligand-gated ion channels that are quite different to Cys-loop and ionotropic glutamate receptors (2-5). P2X receptors are found in many species from several phyla, implying important roles in diverse life forms (6, 7). A key goal is to understand how P2X receptors operate at the chemical level.The first P2X receptor genes were cloned in 1994 (8, 9) leading to important progress over the last decade in understanding how P2X receptors function (10, 11) using biophysical and biochemical approaches. P2X subunits are thus known to possess intracellular N and C termini and two transmembrane (TM) 2 segments separated by a large extracellular loop (12-14). The trimeric architecture of P2X receptors is also well established based on subunit concatemers, blue-native PAGE, and atomic force microscopy experiments (15-18). It is also well established that the P2X pore is lined by TM2 (19 -22), with TM1 making little contribution to ion flow (23, 24). Moreover, careful studies of permeation and selectivity have suggested that the same area of TM2 is responsible for both ion selection and channel opening (25-31). Mutational approaches have shed light on the extracellular domain of P2X receptors, including how the ATP binding pocket may form (32), how the 10 conserved cysteines contribute to the fold of the protein (33, 34), and how cations such as Zn 2ϩ profoundly affect receptor function (35)(36)(37)(38). Finally, meticulous analysis of voltage-jump relaxations for ATP-evoked currents combined with mutagenesis has suggested that TM2 may display hinge-like flexibility (39, 40), and recently the P2X4 receptor open pore has been imaged with fast scanning atomic force microscopy (41). Most of these past studies were conducted on rat P2X2 (rP2X2) receptors and have provided a basis to explore P2X receptor pores and the gating process.In 2009, the field was immeasurably advanced by the landmark achievement and report of a crystal structure of the zebrafish (zf) P2X4.1 receptor in the closed state (42). The P2X receptor topology was similar to that of acid-sensing ion channels (42, 43), a possibility that had been raised by earlier modeling studies (44). However, it is important to note that the extracellular domains of P2X and acid-sensing ion channels are distinct with little sequence or structural homology, whereas the pore domains share similar architectures (43). The availability of direct structural information for P2X receptors substantiated most of the aforementioned mutational studies and for the first time provided a framework to understand their atomic origins (5). Of most relevance here, it directly revealed that the P2X pore was indeed formed by TM2 ␣-helices arranged around a 3-fold molecular axis of symmetry. These were arranged ϳ45°to the membrane normal and crossed about halfway along their l...

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“…Functions of the TM domains in channel gating have been deeply studied, and its roles in pore location, ion permeability and structural rearrangements were confirmed by crystal structures [88][89][90][91][92][93][94][95][96][97] . Under normal conditions, pore opening of P2X receptors is controlled by ATP binding.…”

Section: N-and C-terminimentioning

confidence: 99%

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

Wang

2016

Acta Pharmacol Sin

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P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na + , K + and Ca 2+ . Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATPbound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.

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Thr³³⁹-to-Serine Substitution in Rat P2X₂Receptor Second Transmembrane Domain Causes Constitutive Opening and Indicates a Gating Role for Lys³⁰⁸

Cited by 51 publications

References 39 publications

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

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

Gated Access to the Pore of a P2X Receptor

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

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