Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine
Identifieur interne : 001044 ( Istex/Corpus ); précédent : 001043; suivant : 001045Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine
Auteurs : Rosanne Varkevisser ; Marien J. C. Houtman ; Maaike Waasdorp ; Joyce C. K. Man ; Raimond Heukers ; Hiroki Takanari ; Ralph G. Tieland ; Paul M. P. Van Bergen En Henegouwen ; Marc A. Vos ; Marcel A. G. Van Der HeydenSource :
- Pflügers Archiv - European Journal of Physiology [ 0031-6768 ] ; 2013-02-01.
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Abstract
Abstract: Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased KIR2.x carried IK1 current and that inhibiting protein degradation in the lysosome increased intracellular KIR2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of KIR2.1 to further restore normal levels of functional KIR2.1 channels. KIR2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). KIR2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved KIR2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased IK1 (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute IK1 block. Baf and CQ did not significantly enhance IK1 densities. Pentamidine (10 μM, 48 h) reduced KIR2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in KIR2.1 degradation. Pentamidine-induced downregulation of KIR2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.
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DOI: 10.1007/s00424-012-1189-5
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C." last="Houtman">Marien J. C. Houtman</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Waasdorp, Maaike" sort="Waasdorp, Maaike" uniqKey="Waasdorp M" first="Maaike" last="Waasdorp">Maaike Waasdorp</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Man, Joyce C K" sort="Man, Joyce C K" uniqKey="Man J" first="Joyce C. K." last="Man">Joyce C. K. Man</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Heukers, Raimond" sort="Heukers, Raimond" uniqKey="Heukers R" first="Raimond" last="Heukers">Raimond Heukers</name><affiliation><mods:affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Takanari, Hiroki" sort="Takanari, Hiroki" uniqKey="Takanari H" first="Hiroki" last="Takanari">Hiroki Takanari</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Tieland, Ralph G" sort="Tieland, Ralph G" uniqKey="Tieland R" first="Ralph G." last="Tieland">Ralph G. Tieland</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Van Bergen En Henegouwen, Paul M P" sort="Van Bergen En Henegouwen, Paul M P" uniqKey="Van Bergen En Henegouwen P" first="Paul M. P." last="Van Bergen En Henegouwen">Paul M. P. Van Bergen En Henegouwen</name><affiliation><mods:affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Vos, Marc A" sort="Vos, Marc A" uniqKey="Vos M" first="Marc A." last="Vos">Marc A. Vos</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation></author><author><name sortKey="Van Der Heyden, Marcel A G" sort="Van Der Heyden, Marcel A G" uniqKey="Van Der Heyden M" first="Marcel A. G." last="Van Der Heyden">Marcel A. G. Van Der Heyden</name><affiliation><mods:affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: m.a.g.vanderheyden@umcutrecht.nl</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Pflügers Archiv - European Journal of Physiology</title><title level="j" type="sub">European Journal of Physiology</title><title level="j" type="abbrev">Pflugers Arch - Eur J Physiol</title><idno type="ISSN">0031-6768</idno><idno type="eISSN">1432-2013</idno><imprint><publisher>Springer-Verlag</publisher><pubPlace>Berlin/Heidelberg</pubPlace><date type="published" when="2013-02-01">2013-02-01</date><biblScope unit="volume">465</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="247">247</biblScope><biblScope unit="page" to="259">259</biblScope></imprint><idno type="ISSN">0031-6768</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0031-6768</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chloroquine</term><term>Chlorpromazine</term><term>Dynamin</term><term>Internalization</term><term>Inward rectifier</term><term>KIR2.1</term><term>Pentamidine</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased KIR2.x carried IK1 current and that inhibiting protein degradation in the lysosome increased intracellular KIR2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of KIR2.1 to further restore normal levels of functional KIR2.1 channels. KIR2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). KIR2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved KIR2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased IK1 (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute IK1 block. Baf and CQ did not significantly enhance IK1 densities. Pentamidine (10 μM, 48 h) reduced KIR2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in KIR2.1 degradation. Pentamidine-induced downregulation of KIR2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.</div></front></TEI><istex><corpusName>springer-journals</corpusName><author><json:item><name>Rosanne Varkevisser</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Marien J. C. Houtman</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Maaike Waasdorp</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Joyce C. K. Man</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Raimond Heukers</name><affiliations><json:string>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Hiroki Takanari</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Ralph G. Tieland</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Paul M. P. van Bergen en Henegouwen</name><affiliations><json:string>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Marc A. Vos</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string></affiliations></json:item><json:item><name>Marcel A. G. van der Heyden</name><affiliations><json:string>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</json:string><json:string>E-mail: m.a.g.vanderheyden@umcutrecht.nl</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Inward rectifier</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>KIR2.1</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Internalization</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Dynamin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Pentamidine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Chlorpromazine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Chloroquine</value></json:item></subject><articleId><json:string>1189</json:string><json:string>s00424-012-1189-5</json:string></articleId><arkIstex>ark:/67375/VQC-03CXQNBZ-K</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>OriginalPaper</json:string></originalGenre><abstract>Abstract: Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased KIR2.x carried IK1 current and that inhibiting protein degradation in the lysosome increased intracellular KIR2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of KIR2.1 to further restore normal levels of functional KIR2.1 channels. KIR2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). KIR2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved KIR2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased IK1 (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p > 0.05), while the CPZ effect on current density was not testable due to acute IK1 block. Baf and CQ did not significantly enhance IK1 densities. Pentamidine (10 μM, 48 h) reduced KIR2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in KIR2.1 degradation. Pentamidine-induced downregulation of KIR2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.</abstract><qualityIndicators><score>9.7</score><pdfWordCount>7038</pdfWordCount><pdfCharCount>44059</pdfCharCount><pdfVersion>1.4</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>595.276 x 790.866 pts</pdfPageSize><refBibsNative>false</refBibsNative><abstractWordCount>225</abstractWordCount><abstractCharCount>1680</abstractCharCount><keywordCount>7</keywordCount></qualityIndicators><title>Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine</title><genre><json:string>research-article</json:string></genre><host><title>Pflügers Archiv - European Journal of 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type="first">Rosanne</forename><surname>Varkevisser</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Marien</forename><surname>Houtman</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Maaike</forename><surname>Waasdorp</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Joyce</forename><surname>Man</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Raimond</forename><surname>Heukers</surname></persName><affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Hiroki</forename><surname>Takanari</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Ralph</forename><surname>Tieland</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Paul</forename><surname>van Bergen en Henegouwen</surname></persName><affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0008"><persName><forename type="first">Marc</forename><surname>Vos</surname></persName><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><author xml:id="author-0009" corresp="yes"><persName><forename type="first">Marcel</forename><surname>van der Heyden</surname></persName><email>m.a.g.vanderheyden@umcutrecht.nl</email><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation></author><idno type="istex">95C9BA075D70387B12EA4063E576F6F84B86A84F</idno><idno type="ark">ark:/67375/VQC-03CXQNBZ-K</idno><idno type="DOI">10.1007/s00424-012-1189-5</idno><idno type="article-id">1189</idno><idno type="article-id">s00424-012-1189-5</idno></analytic><monogr><title level="j">Pflügers Archiv - European Journal of Physiology</title><title level="j" type="sub">European Journal of Physiology</title><title level="j" type="abbrev">Pflugers Arch - Eur J Physiol</title><idno type="pISSN">0031-6768</idno><idno type="eISSN">1432-2013</idno><idno type="journal-ID">true</idno><idno type="issue-article-count">14</idno><idno type="volume-issue-count">12</idno><imprint><publisher>Springer-Verlag</publisher><pubPlace>Berlin/Heidelberg</pubPlace><date type="published" when="2013-02-01"></date><biblScope unit="volume">465</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="247">247</biblScope><biblScope unit="page" to="259">259</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2012-08-29</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased KIR2.x carried IK1 current and that inhibiting protein degradation in the lysosome increased intracellular KIR2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of KIR2.1 to further restore normal levels of functional KIR2.1 channels. KIR2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). KIR2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved KIR2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased IK1 (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute IK1 block. Baf and CQ did not significantly enhance IK1 densities. Pentamidine (10 μM, 48 h) reduced KIR2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in KIR2.1 degradation. Pentamidine-induced downregulation of KIR2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Inward rectifier</term></item><item><term>KIR2.1</term></item><item><term>Internalization</term></item><item><term>Dynamin</term></item><item><term>Pentamidine</term></item><item><term>Chlorpromazine</term></item><item><term>Chloroquine</term></item></list></keywords></textClass><textClass><keywords scheme="Journal-Subject-Collection"><list><label>SC3</label><item><term>Biomedical and Life Sciences</term></item></list></keywords></textClass><textClass><keywords scheme="Journal-Subject-Group"><list><label>SCB</label><item><term>Biomedicine</term></item><label>SCB13004</label><item><term>Human Physiology</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2012-08-29">Created</change><change when="2013-02-01">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/VQC-03CXQNBZ-K/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="corpus springer-journals not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//Springer-Verlag//DTD A++ V2.4//EN" URI="http://devel.springer.de/A++/V2.4/DTD/A++V2.4.dtd" name="istex:docType"></istex:docType><istex:document><Publisher><PublisherInfo><PublisherName>Springer-Verlag</PublisherName><PublisherLocation>Berlin/Heidelberg</PublisherLocation><PublisherImprintName>Springer</PublisherImprintName></PublisherInfo><Journal OutputMedium="All"><JournalInfo JournalProductType="ArchiveJournal" NumberingStyle="Unnumbered"><JournalID>424</JournalID><JournalPrintISSN>0031-6768</JournalPrintISSN><JournalElectronicISSN>1432-2013</JournalElectronicISSN><JournalTitle>Pflügers Archiv - European Journal of Physiology</JournalTitle><JournalSubTitle>European Journal of Physiology</JournalSubTitle><JournalAbbreviatedTitle>Pflugers Arch - Eur J Physiol</JournalAbbreviatedTitle><JournalSubjectGroup><JournalSubject Code="SCB" Type="Primary">Biomedicine</JournalSubject><JournalSubject Code="SCB13004" Priority="1" Type="Secondary">Human Physiology</JournalSubject><SubjectCollection Code="SC3">Biomedical and Life Sciences</SubjectCollection></JournalSubjectGroup></JournalInfo><Volume OutputMedium="All"><VolumeInfo TocLevels="0" VolumeType="Regular"><VolumeIDStart>465</VolumeIDStart><VolumeIDEnd>465</VolumeIDEnd><VolumeIssueCount>12</VolumeIssueCount></VolumeInfo><Issue IssueType="Regular" OutputMedium="All"><IssueInfo IssueType="Regular" TocLevels="0"><IssueIDStart>2</IssueIDStart><IssueIDEnd>2</IssueIDEnd><IssueArticleCount>14</IssueArticleCount><IssueHistory><OnlineDate><Year>2013</Year><Month>1</Month><Day>31</Day></OnlineDate><PrintDate><Year>2013</Year><Month>1</Month><Day>30</Day></PrintDate><CoverDate><Year>2013</Year><Month>2</Month></CoverDate><PricelistYear>2013</PricelistYear></IssueHistory><IssueCopyright><CopyrightHolderName>Springer-Verlag Berlin Heidelberg</CopyrightHolderName><CopyrightYear>2013</CopyrightYear></IssueCopyright></IssueInfo><Article ID="s00424-012-1189-5" OutputMedium="All"><ArticleInfo ArticleType="OriginalPaper" ContainsESM="No" Language="En" NumberingStyle="Unnumbered" TocLevels="0"><ArticleID>1189</ArticleID><ArticleDOI>10.1007/s00424-012-1189-5</ArticleDOI><ArticleSequenceNumber>7</ArticleSequenceNumber><ArticleTitle Language="En">Inhibiting the clathrin-mediated endocytosis pathway rescues K<Subscript>IR</Subscript>2.1 downregulation by pentamidine</ArticleTitle><ArticleCategory>Ion Channels, Receptors and Transporters</ArticleCategory><ArticleFirstPage>247</ArticleFirstPage><ArticleLastPage>259</ArticleLastPage><ArticleHistory><RegistrationDate><Year>2012</Year><Month>11</Month><Day>14</Day></RegistrationDate><Received><Year>2012</Year><Month>8</Month><Day>29</Day></Received><Revised><Year>2012</Year><Month>11</Month><Day>9</Day></Revised><Accepted><Year>2012</Year><Month>11</Month><Day>14</Day></Accepted><OnlineDate><Year>2012</Year><Month>11</Month><Day>29</Day></OnlineDate></ArticleHistory><ArticleCopyright><CopyrightHolderName>Springer-Verlag Berlin Heidelberg</CopyrightHolderName><CopyrightYear>2012</CopyrightYear></ArticleCopyright><ArticleGrants Type="Regular"><MetadataGrant Grant="OpenAccess"></MetadataGrant><AbstractGrant Grant="OpenAccess"></AbstractGrant><BodyPDFGrant Grant="Restricted"></BodyPDFGrant><BodyHTMLGrant Grant="Restricted"></BodyHTMLGrant><BibliographyGrant Grant="Restricted"></BibliographyGrant><ESMGrant Grant="Restricted"></ESMGrant></ArticleGrants></ArticleInfo><ArticleHeader><AuthorGroup><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Rosanne</GivenName><FamilyName>Varkevisser</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Marien</GivenName><GivenName>J.</GivenName><GivenName>C.</GivenName><FamilyName>Houtman</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Maaike</GivenName><FamilyName>Waasdorp</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Joyce</GivenName><GivenName>C.</GivenName><GivenName>K.</GivenName><FamilyName>Man</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Raimond</GivenName><FamilyName>Heukers</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Hiroki</GivenName><FamilyName>Takanari</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Ralph</GivenName><GivenName>G.</GivenName><FamilyName>Tieland</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Paul</GivenName><GivenName>M.</GivenName><GivenName>P.</GivenName><Particle>van</Particle><FamilyName>Bergen en Henegouwen</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Marc</GivenName><GivenName>A.</GivenName><FamilyName>Vos</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1" CorrespondingAffiliationID="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Marcel</GivenName><GivenName>A.</GivenName><GivenName>G.</GivenName><Particle>van der</Particle><FamilyName>Heyden</FamilyName></AuthorName><Contact><Phone>+31-30-2538900</Phone><Fax>+31-30-2539036</Fax><Email>m.a.g.vanderheyden@umcutrecht.nl</Email></Contact></Author><Affiliation ID="Aff1"><OrgDivision>Department of Medical Physiology, Division of Heart & Lungs</OrgDivision><OrgName>UMCU</OrgName><OrgAddress><Street>Yalelaan 50</Street><Postcode>3584 CM</Postcode><City>Utrecht</City><Country Code="NL">The Netherlands</Country></OrgAddress></Affiliation><Affiliation ID="Aff2"><OrgDivision>Cell Biology, Department of Biology, Science Faculty</OrgDivision><OrgName>Utrecht University</OrgName><OrgAddress><City>Utrecht</City><Country Code="NL">The Netherlands</Country></OrgAddress></Affiliation></AuthorGroup><Abstract ID="Abs1" Language="En" OutputMedium="All"><Heading>Abstract</Heading><Para>Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K<Subscript>IR</Subscript>2.x carried I<Subscript>K1</Subscript> current and that inhibiting protein degradation in the lysosome increased intracellular K<Subscript>IR</Subscript>2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K<Subscript>IR</Subscript>2.1 to further restore normal levels of functional K<Subscript>IR</Subscript>2.1 channels. K<Subscript>IR</Subscript>2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K<Subscript>IR</Subscript>2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K<Subscript>IR</Subscript>2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I<Subscript>K1</Subscript> (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (<Emphasis Type="Italic">N</Emphasis> = 9 and 13 respectively, <Emphasis Type="Italic">p</Emphasis> < 0.05), while the CPZ effect on current density was not testable due to acute I<Subscript>K1</Subscript> block. Baf and CQ did not significantly enhance I<Subscript>K1</Subscript> densities. Pentamidine (10 μM, 48 h) reduced K<Subscript>IR</Subscript>2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K<Subscript>IR</Subscript>2.1 degradation. Pentamidine-induced downregulation of K<Subscript>IR</Subscript>2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.</Para></Abstract><KeywordGroup Language="En" OutputMedium="All"><Heading>Keywords</Heading><Keyword>Inward rectifier</Keyword><Keyword>K<Subscript>IR</Subscript>2.1</Keyword><Keyword>Internalization</Keyword><Keyword>Dynamin</Keyword><Keyword>Pentamidine</Keyword><Keyword>Chlorpromazine</Keyword><Keyword>Chloroquine</Keyword></KeywordGroup><ArticleNote Type="Misc"><SimplePara>Rosanne Varkevisser and Marien JC Houtman contributed equally to this work.</SimplePara></ArticleNote></ArticleHeader><NoBody></NoBody></Article></Issue></Volume></Journal></Publisher></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine</title></titleInfo><name type="personal"><namePart type="given">Rosanne</namePart><namePart type="family">Varkevisser</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Marien</namePart><namePart type="given">J.</namePart><namePart type="given">C.</namePart><namePart type="family">Houtman</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Maaike</namePart><namePart type="family">Waasdorp</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Joyce</namePart><namePart type="given">C.</namePart><namePart type="given">K.</namePart><namePart type="family">Man</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Raimond</namePart><namePart type="family">Heukers</namePart><affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hiroki</namePart><namePart type="family">Takanari</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Ralph</namePart><namePart type="given">G.</namePart><namePart type="family">Tieland</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Paul</namePart><namePart type="given">M.</namePart><namePart type="given">P.</namePart><namePart type="family">van Bergen en Henegouwen</namePart><affiliation>Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Marc</namePart><namePart type="given">A.</namePart><namePart type="family">Vos</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="given">Marcel</namePart><namePart type="given">A.</namePart><namePart type="given">G.</namePart><namePart type="family">van der Heyden</namePart><affiliation>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands</affiliation><affiliation>E-mail: m.a.g.vanderheyden@umcutrecht.nl</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="OriginalPaper" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>Springer-Verlag</publisher><place><placeTerm type="text">Berlin/Heidelberg</placeTerm></place><dateCreated encoding="w3cdtf">2012-08-29</dateCreated><dateIssued encoding="w3cdtf">2013-02-01</dateIssued><copyrightDate encoding="w3cdtf">2012</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><abstract lang="en">Abstract: Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased KIR2.x carried IK1 current and that inhibiting protein degradation in the lysosome increased intracellular KIR2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of KIR2.1 to further restore normal levels of functional KIR2.1 channels. KIR2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). KIR2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved KIR2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased IK1 (at −60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute IK1 block. Baf and CQ did not significantly enhance IK1 densities. Pentamidine (10 μM, 48 h) reduced KIR2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in KIR2.1 degradation. Pentamidine-induced downregulation of KIR2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.</abstract><note>Ion Channels, Receptors and Transporters</note><subject lang="en"><genre>Keywords</genre><topic>Inward rectifier</topic><topic>KIR2.1</topic><topic>Internalization</topic><topic>Dynamin</topic><topic>Pentamidine</topic><topic>Chlorpromazine</topic><topic>Chloroquine</topic></subject><relatedItem type="host"><titleInfo><title>Pflügers Archiv - European Journal of Physiology</title><subTitle>European Journal of Physiology</subTitle></titleInfo><titleInfo type="abbreviated"><title>Pflugers Arch - Eur J Physiol</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>Springer</publisher><dateIssued encoding="w3cdtf">2013-01-31</dateIssued><copyrightDate encoding="w3cdtf">2013</copyrightDate></originInfo><subject><genre>Journal-Subject-Collection</genre><topic authority="SpringerSubjectCodes" authorityURI="SC3">Biomedical and Life Sciences</topic></subject><subject><genre>Journal-Subject-Group</genre><topic authority="SpringerSubjectCodes" authorityURI="SCB">Biomedicine</topic><topic authority="SpringerSubjectCodes" authorityURI="SCB13004">Human Physiology</topic></subject><identifier type="ISSN">0031-6768</identifier><identifier type="eISSN">1432-2013</identifier><identifier type="JournalID">424</identifier><identifier type="IssueArticleCount">14</identifier><identifier type="VolumeIssueCount">12</identifier><part><date>2013</date><detail type="volume"><number>465</number><caption>vol.</caption></detail><detail type="issue"><number>2</number><caption>no.</caption></detail><extent unit="pages"><start>247</start><end>259</end></extent></part><recordInfo><recordOrigin>Springer-Verlag Berlin Heidelberg, 2013</recordOrigin></recordInfo></relatedItem><identifier type="istex">95C9BA075D70387B12EA4063E576F6F84B86A84F</identifier><identifier type="ark">ark:/67375/VQC-03CXQNBZ-K</identifier><identifier type="DOI">10.1007/s00424-012-1189-5</identifier><identifier type="ArticleID">1189</identifier><identifier type="ArticleID">s00424-012-1189-5</identifier><accessCondition type="use and reproduction" contentType="copyright">Springer-Verlag Berlin Heidelberg, 2012</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-3XSW68JL-F">springer</recordContentSource><recordOrigin>Springer-Verlag Berlin Heidelberg, 2012</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/VQC-03CXQNBZ-K/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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