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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor</title><author><name sortKey="Lorinczi, Eva" sort="Lorinczi, Eva" uniqKey="Lorinczi E" first="Éva" last="Lörinczi">Éva Lörinczi</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Bhargava, Yogesh" sort="Bhargava, Yogesh" uniqKey="Bhargava Y" first="Yogesh" last="Bhargava">Yogesh Bhargava</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Marino, Stephen F" sort="Marino, Stephen F" uniqKey="Marino S" first="Stephen F." last="Marino">Stephen F. Marino</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Neurochemistry,<institution>Max Planck Institute for Brain Research</institution>, 60528 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Taly, Antoine" sort="Taly, Antoine" uniqKey="Taly A" first="Antoine" last="Taly">Antoine Taly</name><affiliation><nlm:aff wicri:cut="; and" id="aff4">Laboratoire de Biophysicochimie des Récepteurs Canaux,<institution>Unité Mixte de Recherche 7199 Faculté de Pharmacie—Université de Strasbourg</institution>, 67401 Illkirch,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Kaczmarek Hajek, Karina" sort="Kaczmarek Hajek, Karina" uniqKey="Kaczmarek Hajek K" first="Karina" last="Kaczmarek-Hájek">Karina Kaczmarek-Hájek</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Barrantes Freer, Alonso" sort="Barrantes Freer, Alonso" uniqKey="Barrantes Freer A" first="Alonso" last="Barrantes-Freer">Alonso Barrantes-Freer</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Dutertre, Sebastien" sort="Dutertre, Sebastien" uniqKey="Dutertre S" first="Sébastien" last="Dutertre">Sébastien Dutertre</name><affiliation><nlm:aff id="aff5">Institute for Molecular Bioscience,<institution>University of Queensland</institution>, Brisbane QLD 4072,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Grutter, Thomas" sort="Grutter, Thomas" uniqKey="Grutter T" first="Thomas" last="Grutter">Thomas Grutter</name><affiliation><nlm:aff wicri:cut="; and" id="aff4">Laboratoire de Biophysicochimie des Récepteurs Canaux,<institution>Unité Mixte de Recherche 7199 Faculté de Pharmacie—Université de Strasbourg</institution>, 67401 Illkirch,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Rettinger, Jurgen" sort="Rettinger, Jurgen" uniqKey="Rettinger J" first="Jürgen" last="Rettinger">Jürgen Rettinger</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Nicke, Annette" sort="Nicke, Annette" uniqKey="Nicke A" first="Annette" last="Nicke">Annette Nicke</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Neurochemistry,<institution>Max Planck Institute for Brain Research</institution>, 60528 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22745172</idno><idno type="pmc">3396496</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396496</idno><idno type="RBID">PMC:3396496</idno><idno type="doi">10.1073/pnas.1118759109</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">001B69</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001B69</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor</title><author><name sortKey="Lorinczi, Eva" sort="Lorinczi, Eva" uniqKey="Lorinczi E" first="Éva" last="Lörinczi">Éva Lörinczi</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Bhargava, Yogesh" sort="Bhargava, Yogesh" uniqKey="Bhargava Y" first="Yogesh" last="Bhargava">Yogesh Bhargava</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Marino, Stephen F" sort="Marino, Stephen F" uniqKey="Marino S" first="Stephen F." last="Marino">Stephen F. Marino</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Neurochemistry,<institution>Max Planck Institute for Brain Research</institution>, 60528 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Taly, Antoine" sort="Taly, Antoine" uniqKey="Taly A" first="Antoine" last="Taly">Antoine Taly</name><affiliation><nlm:aff wicri:cut="; and" id="aff4">Laboratoire de Biophysicochimie des Récepteurs Canaux,<institution>Unité Mixte de Recherche 7199 Faculté de Pharmacie—Université de Strasbourg</institution>, 67401 Illkirch,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Kaczmarek Hajek, Karina" sort="Kaczmarek Hajek, Karina" uniqKey="Kaczmarek Hajek K" first="Karina" last="Kaczmarek-Hájek">Karina Kaczmarek-Hájek</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Barrantes Freer, Alonso" sort="Barrantes Freer, Alonso" uniqKey="Barrantes Freer A" first="Alonso" last="Barrantes-Freer">Alonso Barrantes-Freer</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Dutertre, Sebastien" sort="Dutertre, Sebastien" uniqKey="Dutertre S" first="Sébastien" last="Dutertre">Sébastien Dutertre</name><affiliation><nlm:aff id="aff5">Institute for Molecular Bioscience,<institution>University of Queensland</institution>, Brisbane QLD 4072,<country>Australia</country></nlm:aff></affiliation></author><author><name sortKey="Grutter, Thomas" sort="Grutter, Thomas" uniqKey="Grutter T" first="Thomas" last="Grutter">Thomas Grutter</name><affiliation><nlm:aff wicri:cut="; and" id="aff4">Laboratoire de Biophysicochimie des Récepteurs Canaux,<institution>Unité Mixte de Recherche 7199 Faculté de Pharmacie—Université de Strasbourg</institution>, 67401 Illkirch,<country>France</country></nlm:aff></affiliation></author><author><name sortKey="Rettinger, Jurgen" sort="Rettinger, Jurgen" uniqKey="Rettinger J" first="Jürgen" last="Rettinger">Jürgen Rettinger</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author><author><name sortKey="Nicke, Annette" sort="Nicke, Annette" uniqKey="Nicke A" first="Annette" last="Nicke">Annette Nicke</name><affiliation><nlm:aff id="aff1">Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Neurochemistry,<institution>Max Planck Institute for Brain Research</institution>, 60528 Frankfurt,<country>Germany</country>;</nlm:aff></affiliation></author></analytic><series><title level="j">Proceedings of the National Academy of Sciences of the United States of America</title><idno type="ISSN">0027-8424</idno><idno type="eISSN">1091-6490</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120–I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-<italic>O</italic>-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP–induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Proc Natl Acad Sci U S A</journal-id><journal-id journal-id-type="iso-abbrev">Proc. Natl. Acad. Sci. U.S.A</journal-id><journal-id journal-id-type="hwp">pnas</journal-id><journal-id journal-id-type="pmc">pnas</journal-id><journal-id journal-id-type="publisher-id">PNAS</journal-id><journal-title-group><journal-title>Proceedings of the National Academy of Sciences of the United States of America</journal-title></journal-title-group><issn pub-type="ppub">0027-8424</issn><issn pub-type="epub">1091-6490</issn><publisher><publisher-name>National Academy of Sciences</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22745172</article-id><article-id pub-id-type="pmc">3396496</article-id><article-id pub-id-type="publisher-id">201118759</article-id><article-id pub-id-type="doi">10.1073/pnas.1118759109</article-id><article-categories><subj-group subj-group-type="heading"><subject>Biological Sciences</subject><subj-group><subject>Pharmacology</subject></subj-group></subj-group></article-categories><title-group><article-title>Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor</article-title><alt-title alt-title-type="short">Voltage clamp fluorometry on P2X1 receptors</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lörinczi</surname><given-names>Éva</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref><xref ref-type="author-notes" rid="fn1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bhargava</surname><given-names>Yogesh</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref><xref ref-type="author-notes" rid="fn2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Marino</surname><given-names>Stephen F.</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref><xref ref-type="aff" rid="aff3"><sup>c</sup></xref><xref ref-type="author-notes" rid="fn2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Taly</surname><given-names>Antoine</given-names></name><xref ref-type="aff" rid="aff4"><sup>d</sup></xref><xref ref-type="author-notes" rid="fn2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kaczmarek-Hájek</surname><given-names>Karina</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Barrantes-Freer</surname><given-names>Alonso</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Dutertre</surname><given-names>Sébastien</given-names></name><xref ref-type="aff" rid="aff5"><sup>e</sup></xref></contrib><contrib contrib-type="author"><name><surname>Grutter</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="aff4"><sup>d</sup></xref></contrib><contrib contrib-type="author"><name><surname>Rettinger</surname><given-names>Jürgen</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Nicke</surname><given-names>Annette</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref><xref ref-type="aff" rid="aff3"><sup>c</sup></xref><xref ref-type="corresp" rid="cor1"><sup>4</sup></xref></contrib><aff id="aff1"><sup>a</sup>Department of Molecular Biology of Neuronal Signals,<institution>Max Planck Institute for Experimental Medicine</institution>, 37075 Göttingen,<country>Germany</country>;</aff><aff id="aff2"><sup>b</sup>Department of Biophysical Chemistry,<institution>Max Planck Institute for Biophysics</institution>, 60438 Frankfurt,<country>Germany</country>;</aff><aff id="aff3"><sup>c</sup>Department of Neurochemistry,<institution>Max Planck Institute for Brain Research</institution>, 60528 Frankfurt,<country>Germany</country>;</aff><aff id="aff4"><sup>d</sup>Laboratoire de Biophysicochimie des Récepteurs Canaux,<institution>Unité Mixte de Recherche 7199 Faculté de Pharmacie—Université de Strasbourg</institution>, 67401 Illkirch,<country>France</country>; and</aff><aff id="aff5"><sup>e</sup>Institute for Molecular Bioscience,<institution>University of Queensland</institution>, Brisbane QLD 4072,<country>Australia</country></aff></contrib-group><author-notes><corresp id="cor1"><sup>4</sup>To whom correspondence should be addressed. E-mail: <email>anicke@gwdg.de</email>.</corresp><fn fn-type="edited-by"><p>Edited by Eric Gouaux, Oregon Health & Science University, Portland, OR, and approved May 18, 2012 (received for review November 28, 2011)</p></fn><fn fn-type="con"><p>Author contributions: T.G., J.R., and A.N. designed research; É.L., Y.B., S.F.M., A.T., K.K.-H., A.B.-F., S.D., and A.N. performed research; É.L., S.F.M., A.T., K.K.-H., A.B.-F., S.D., J.R., and A.N. analyzed data; and É.L., J.R., and A.N. wrote the paper.</p></fn><fn fn-type="equal" id="fn2"><p><sup>2</sup>Y.B., S.F.M., and A.T. contributed equally to this work.</p></fn><fn fn-type="present-address" id="fn1"><p><sup>1</sup>Present address: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.</p></fn><fn fn-type="present-address" id="fn3"><p><sup>3</sup>Present address: Department of Crystallography, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.</p></fn></author-notes><pub-date pub-type="ppub"><day>10</day><month>7</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>27</day><month>6</month><year>2012</year></pub-date><volume>109</volume><issue>28</issue><fpage>11396</fpage><lpage>11401</lpage><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="pnas.201118759.pdf"></self-uri><abstract><p>P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120–I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-<italic>O</italic>-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP–induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.</p></abstract></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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