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Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine

  • Ion Channels, Receptors and Transporters
  • Published:
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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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Acknowledgments

The research leading to the results has received funding from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. HEALTH-F2-2009-241526, EUTrigTreat.

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Authors and Affiliations

  1. Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan 50, 3584 CM, Utrecht, The Netherlands

    Rosanne Varkevisser, Marien J. C. Houtman, Maaike Waasdorp, Joyce C. K. Man, Hiroki Takanari, Ralph G. Tieland, Marc A. Vos & Marcel A. G. van der Heyden

  2. Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands

    Raimond Heukers & Paul M. P. van Bergen en Henegouwen

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  1. Rosanne Varkevisser
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  2. Marien J. C. Houtman
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  3. Maaike Waasdorp
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Correspondence to Marcel A. G. van der Heyden.

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Rosanne Varkevisser and Marien JC Houtman contributed equally to this work.

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Varkevisser, R., Houtman, M.J.C., Waasdorp, M. et al. Inhibiting the clathrin-mediated endocytosis pathway rescues KIR2.1 downregulation by pentamidine. Pflugers Arch - Eur J Physiol 465, 247–259 (2013). https://doi.org/10.1007/s00424-012-1189-5

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