Inhibiting the clathrin-mediated endocytosis pathway rescues K IR2.1 downregulation by pentamidine

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 Heyden*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K IR2.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). K IR2.1 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 I K1 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.

Original languageEnglish
Pages (from-to)247-259
Number of pages13
JournalPflugers Archiv European Journal of Physiology
Volume465
Issue number2
DOIs
Publication statusPublished - 1 Feb 2013
Externally publishedYes

Cite this

Varkevisser, R., Houtman, M. J. C., Waasdorp, M., Man, J. C. K., Heukers, R., Takanari, H., ... Van Der Heyden, M. A. G. (2013). Inhibiting the clathrin-mediated endocytosis pathway rescues K IR2.1 downregulation by pentamidine. Pflugers Archiv European Journal of Physiology, 465(2), 247-259. https://doi.org/10.1007/s00424-012-1189-5