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

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.