Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine

Pflugers Arch. 2013 Feb;465(2):247-59. doi: 10.1007/s00424-012-1189-5. Epub 2012 Nov 29.

Abstract

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K(IR)2.x carried I(K1) current and that inhibiting protein degradation in the lysosome increased intracellular K(IR)2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K(IR)2.1 to further restore normal levels of functional K(IR)2.1 channels. K(IR)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(IR)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(IR)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(K1) (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 I(K1) densities. Pentamidine (10 μM, 48 h) reduced K(IR)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(IR)2.1 degradation. Pentamidine-induced downregulation of K(IR)2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Antiprotozoal Agents / pharmacology*
  • Cell Membrane / metabolism
  • Chloroquine / pharmacology
  • Chlorpromazine / pharmacology
  • Clathrin-Coated Vesicles / metabolism*
  • Down-Regulation*
  • Endocytosis / drug effects*
  • HEK293 Cells
  • Humans
  • Hydrazones / pharmacology
  • Macrolides / pharmacology
  • Pentamidine / pharmacology*
  • Potassium Channels, Inwardly Rectifying / drug effects
  • Potassium Channels, Inwardly Rectifying / metabolism*
  • Potassium Channels, Inwardly Rectifying / physiology
  • Protein Transport / drug effects
  • Proteolysis

Substances

  • Antiprotozoal Agents
  • Hydrazones
  • Kir2.1 channel
  • Macrolides
  • N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide
  • Potassium Channels, Inwardly Rectifying
  • Pentamidine
  • Chloroquine
  • bafilomycin A1
  • Chlorpromazine