Regulation of the Cardiac Iks Channel Complex by Ubiquitylation and De-Ubiquitylation

KCNQ1 and its β-subunit KCNE1 form the delayed rectifier potassium current IKs, playing an important role in repolarisation of the cardiac tissue and in water and salt transport across epithelial tissues. In the heart IKs is partly responsible for terminating the cardiac action potential. Malfunctions in this channel can result in arrhythmias leading to cardiac arrest.In heart physiology proper function and regulation of IKs current is essential. It has been reported that one of the mechanisms controlling the membrane density of KCNQ1 channels is mediated by ubiquitylation. IKs was shown to be down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases and this interaction was dependent on the PY-motif on the C-terminal of KCNQ1. Recently it was also discovered that epithelial sodium channel ENaC is regulated by the reverse process - de-ubiquitylation, mediated by an enzyme USP2 (ubiquitin-specific protease 2), which is one of the best described de-ubiquitylases. Therefore the aim of the work was to investigate whether a similar mechanism is valid for KCNQ1/E1 channel complex.The effect of USP2-mediated de-ubiquitylation on IKs channel was investigated using electrophysiology and biochemistry. We observed that when KCNQ1/E1 was co-expressed with USP2-45 or USP2-69 isoform and Nedd4-2 in oocytes, USP2 counteracted the Nedd4-2-specific down-regulation of IKs. It resulted in a rescue of the current amplitude, which was then comparable to the one of IKs expressed alone. Biochemical studies of transfected HEK293 cells confirmed this observation as both total and surface expressed KCNQ1 protein was more abundant when co-expressed with USP2-45/-69 and Nedd4-2 as compared to Nedd4-2 alone. Co-immunoprecipitation assay suggested that USP2 can bind to KCNQ1 independently of the PY-motif and the presence of Nedd4-2.These results point towards an interplay between ubiquitylating enzymes and de-ubiquitylases acting on IKs channel complex in vitro.