Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine
Identifieur interne : 001389 ( Main/Exploration ); précédent : 001388; suivant : 001390Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine
Auteurs : Rosanne Varkevisser [Pays-Bas] ; Marien J. C. Houtman [Pays-Bas] ; Maaike Waasdorp [Pays-Bas] ; Joyce C. K. Man [Pays-Bas] ; Raimond Heukers [Pays-Bas] ; Hiroki Takanari [Pays-Bas] ; Ralph G. Tieland [Pays-Bas] ; Paul M. P. Van Bergen En Henegouwen [Pays-Bas] ; Marc A. Vos [Pays-Bas] ; Marcel A. G. Van Der Heyden [Pays-Bas]Source :
- 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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Van Der Heyden</name><affiliation wicri:level="3"><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht</wicri:regionArea><placeName><settlement type="city">Utrecht</settlement><region nuts="2" type="province">Utrecht (province)</region></placeName></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Pays-Bas</country></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><affiliations><list><country><li>Pays-Bas</li></country><region><li>Utrecht (province)</li></region><settlement><li>Utrecht</li></settlement><orgName><li>Université d'Utrecht</li></orgName></list><tree><country name="Pays-Bas"><region name="Utrecht (province)"><name sortKey="Varkevisser, Rosanne" sort="Varkevisser, Rosanne" uniqKey="Varkevisser R" first="Rosanne" last="Varkevisser">Rosanne Varkevisser</name></region><name sortKey="Heukers, Raimond" sort="Heukers, Raimond" uniqKey="Heukers R" first="Raimond" last="Heukers">Raimond Heukers</name><name sortKey="Houtman, Marien J C" sort="Houtman, Marien J C" uniqKey="Houtman M" first="Marien J. C." last="Houtman">Marien J. C. Houtman</name><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><name sortKey="Takanari, Hiroki" sort="Takanari, Hiroki" uniqKey="Takanari H" first="Hiroki" last="Takanari">Hiroki Takanari</name><name sortKey="Tieland, Ralph G" sort="Tieland, Ralph G" uniqKey="Tieland R" first="Ralph G." last="Tieland">Ralph G. Tieland</name><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><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><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><name sortKey="Vos, Marc A" sort="Vos, Marc A" uniqKey="Vos M" first="Marc A." last="Vos">Marc A. Vos</name><name sortKey="Waasdorp, Maaike" sort="Waasdorp, Maaike" uniqKey="Waasdorp M" first="Maaike" last="Waasdorp">Maaike Waasdorp</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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