title: Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine.
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.
authors.affiliation: Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan, Utrecht, The Netherlands.
authors.forename: Rosanne
authors.initials: R
authors.lastname: Varkevisser
authors.forename: Marien J C
authors.initials: MJ
authors.lastname: Houtman
authors.forename: Maaike
authors.initials: M
authors.lastname: Waasdorp
authors.forename: Joyce C K
authors.initials: JC
authors.lastname: Man
authors.forename: Raimond
authors.initials: R
authors.lastname: Heukers
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takanari
authors.forename: Ralph G
authors.initials: RG
authors.lastname: Tieland
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
authors.forename: Marc A
authors.initials: MA
authors.lastname: Vos
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
mesh_terms: D000200:Action Potentials; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D010419:Pentamidine / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis
chemical_list: D000981:Antiprotozoal Agents; D006835:Hydrazones; C489461:Kir2.1 channel; D018942:Macrolides; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D024661:Potassium Channels, Inwardly Rectifying; D010419:Pentamidine; D002738:Chloroquine; C040929:bafilomycin A1; D002746:Chlorpromazine