Expression and activity of inwardly rectifying potassium (K IR) channels within the heart are strictly regulated. K IR channels have an important role in shaping cardiac action potentials, having a limited conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. Impaired K IR2.1 function causes Andersen-Tawil Syndrome (ATS) and is associated with heart failure. Restoring K IR2.1 function by agonists of K IR2.1 (AgoKirs) would be beneficial. The class 1c antiarrhythmic drug propafenone is identified as an AgoKir; however, its long-term effects on K IR2.1 protein expression, subcellular localization, and function are unknown. Propafenone's long-term effect on K IR2.1 expression and its underlying mechanisms in vitro were investigated. K IR2.1-carried currents were measured by single-cell patch-clamp electrophysiology. K IR2.1 protein expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localization of K IR2.1 proteins. Acute propafenone treatment at low concentrations supports the ability of propafenone to function as an AgoKir without disturbing K IR2.1 protein handling. Chronic propafenone treatment (at 25-100 times higher concentrations than in the acute treatment) increases K IR2.1 protein expression and K IR2.1 current densities in vitro, which are potentially associated with pre-lysosomal trafficking inhibition