Cardiac Potassium Channel hERG Is Regulated by Ubiquitin Ligase Nedd4l

Abstract

The cardiac rapidly activating delayed rectifier potassium channel (IKr) is encoded by the human ether-a-go-go related gene (hERG), which is important for repolarization of the cardiac action potential. Reduction in hERG expression levels due to genetic mutations or drugs causes Long QT Syndrome (LQTS). Recently, we demonstrated that ubiquitination of hERG channels is involved in low K+ induced hERG endocytic degradation. Since homeostatic degradation is an important pathway in maintaining hERG membrane expression levels, we investigated the molecular mechanisms for hERG degradation by focusing on the role and consequence of the ubiquitin (UB) ligase, Nedd4L. Ub plays a role in the internalization of cell-surface hERG channels, and we hypothesized that ubiquitination of hERG channels is facilitated through Nedd4L. To study the effects of Nedd4L on hERG channels, we overexpressed Nedd4L in human embryonic kidney (HEK) 293 cells that stably express the hERG channels. We performed electrophysiological recordings, Western blot, co-immunoprecipitation analysis, and confocal microscopy to identify Nedd4L's role in hERG expression. Our data from whole-cell patch clamp recordings demonstrated that, among hEAG, Kv1.5 and hERG, Nedd4L specifically eliminates the hERG channel current. Western blot and confocal imaging analyses showed that Nedd4L overexpression led to significant reduction in mature hERG channels in the plasma membrane. Data obtained using co-immunoprecipitation indicated that Nedd4L significantly increases ubiquitinated hERG channels. Our data indicate that Nedd4L may play a role in hERG homeostatic degradation. (Supported by the Canadian Institutes of Health Research and Heart and Stroke Foundation of Ontario).