Molecular mechanisms underlying the pilsicainide‐induced stabilization of hERG proteins in transfected mammalian cells

Background Pilsicainide, classified as a relatively selective Na + channel blocker, also has an inhibitory action on the rapidly‐activating delayed‐rectifier K + current ( I Kr ) through human ether‐a‐go‐go‐related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild‐type (WT) hERG proteins and WT‐hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. Methods HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch‐clamp experiments. Results Acute exposure to pilsicainide at 0.03–10 μM influenced neither the expression of WT‐hERG proteins nor WT‐hERG channel currents. Chronic treatment with 0.03–10 μM pilsicainide for 48 h, however, increased the expression of WT‐hERG proteins and channel currents in a concentration‐dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT‐hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane‐impermeant pilsicainide derivative did not influence the expression of WT‐hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF‐1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking‐defective mutant hERG, G601S and R752W. Conclusions Pilsicainide penetrates the plasma membrane, stabilizes WT‐hERG proteins by acting as a chemical chaperone, and enhances WT‐hERG channel currents. This mechanism could also be applicable to modulations of certain mutant‐hERG proteins.