Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG/KCNH2 proteins in the heart.

Reduced levels of the cardiac human (h) ERG ion channel protein and the corresponding repolarizing current I-Kr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Forster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr and disrupted action potential repolarization. Ca2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude and cardiac membrane excitability.