Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

Liang, Xin; Xu, Huijuan; Li, Caiyue; Yin, Shikui; Xu, Tingting; Liu, Jinsong; Li, Zhiyuan

doi:10.1371/journal.pone.0070629

Cited by 8 publications

(8 citation statements)

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“…The present experiments with control concatamers ( Fig. 4 E-H) reveal that the inhibitory bridge forms only when H33 and S345C are present in the same subunit, a finding that is supported further by the demonstration of an inhibitory disulfide bridge between H33C and S345C within individual subunits (16). We therefore conclude that both the potentiating H-C-C and inhibitory H-C metal bridges between TM1 and TM2 occur within individual subunits, consistent with the expectations from the apo and ATP-bound X-ray structures.…”

Section: Biophysical Characterization Of Metal Bridges In Rp2x2 Recepsupporting

confidence: 77%

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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Section: Biophysical Characterization Of Metal Bridges In Rp2x2 Recepsupporting

confidence: 77%

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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Section: Resultscontrasting

confidence: 54%

“…We demonstrated in a few experiments that both the application of H2O2 and the washout of DTT by normal extracellular medium causes a reoxidation of the thiols, resulting in decreased currents ( Figure 3B; cf. Marquez-Klaka et al, 2007;Liang et al, 2013). Figure 3C-E shows the statistical evaluation of all experiments on a percentage basis; as mentioned in the Methods section, data were normalized with respect to the pre-DTT amplitude of the α,β-meATP currents.…”

Section: Reversibility Of Disulfide Bond Formationmentioning

confidence: 99%

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening

Kowalski

Hausmann

Dopychai

et al. 2014

British J Pharmacology

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BACKGROUND AND PURPOSEIt is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACHA molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTSA series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONSIn conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

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“…Finally, this expanding conformation change of the lower body spreads and sets the out ends of the three TM2 helices apart [29] . The resulting relative rotations of TM domains decrease the spaces between TM1 and TM2 domains, thus enlarge the narrowest central pathway of ion conduction and open the pore, which have been further strengthened by our [30] and Swartz' group [31] recent studies upon the zfP2X4 structure.…”

Section: Movements Of the Binding Jaw From Close To Openmentioning

confidence: 56%

ATP Binding and Channel Activation in P2X Receptors

2015

Receptor Clin Invest

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Purinergic P2X receptors are a family of membrane channels activated by extracellular adenosine 5′-triphosphate. They are valuable therapeutic targets primarily for their critical role in neuropathic pain and inflammation. ATP binds allow Na + and Ca 2+ to pass through the open pore, and leads membrane depolarization and an increase of the cytosolic Ca 2+ concentration that initiating Ca 2+ -dependent signaling transduction. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of where ATP binds and how channels gating. The recent publication of the crystal structures of the zebrafish P2X4 receptor adds something new on how P2X receptors work. In this review, we will attempt to present the existing functional data regarding ATP binding with the available crystal structure data and different experimental approaches that have been used to explore the ATP-binding sites.

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Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

Cited by 8 publications

References 50 publications

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

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

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening

ATP Binding and Channel Activation in P2X Receptors

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