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 overexpressing the ubiquitin (Ub) ligase, Nedd4-2 (Neural Precursor Cell- Expressed Developmentally Downregulated Gene 4- 2) (Yang & Kumar, 2010). Previous work in the lab demonstrated that Ub plays a role in the internalization of cell-surface hERG channels, and I hypothesized that ubiquitination of hERG channels is facilitated through Nedd4-2. To study the effects of Nedd4-2 on hERG channels, I overexpressed Nedd4-2 in human embryonic kidney (HEK) 293 cells that stably express the hERG channels. Electrophysiological recordings, Western blot, co-immunoprecipitation analysis, and confocal microscopy were performed to identify Nedd4-2’s role in hERG expression. The data from whole-cell patch clamp recordings demonstrated that, among hEAG, Kv1.5 and hERG, Nedd4-2 specifically eliminates the hERG channel current. Western blot and confocal imaging analyses showed that Nedd4-2 overexpression led to a significant reduction in mature hERG channels in the plasma membrane. Data obtained using co-immunoprecipitation indicated that Nedd4-2 significantly increases ubiquitinated hERG channels. These data indicate that Nedd4-2 may play a role in hERG homeostatic degradation. / Thesis (Master, Physiology) -- Queen's University, 2011-08-15 18:17:00.452

The human ether-à-go-go-related gene (hERG) encodes the pore-forming α-subunits of the Kv11.1 channel that is responsible for the cardiac rapidly activating delayed rectifier K+ current (IKr), which plays a critical role in cardiac repolarization. Dysfunction of hERG causes long QT syndrome (LQTS), a cardiac electrical disorder that can lead to severe cardiac arrhythmias and sudden death (Mitcheson et al., 2000a; Roden, 2004; Maier et al., 2006; Misner et al., 2012). The overall function of hERG channels is dependent on the channel density at the plasma membrane as well as proper channel gating. Previous work from our lab demonstrated that degradation of hERG protein in the lysosome is regulated by ubiquitin ligase Nedd4-2-mediated monoubiquitination (Sun et al., 2011; Guo et al., 2012). However, whether the internalized hERG proteins can be recycled back to the plasma membrane remains to be determined. In the present study, we investigated the regulatory effects of various Rabs on hERG channels using Western blot analysis, co-immunoprecipitation (Co-IP), whole-cell patch clamp and immunofluorescence microscopy. The data revealed that, among hERG, human Kv1.5 (cardiac ultra-rapidly activating delayed rectifier K+ channel), and human EAG (ether-à-go-go gene) potassium channels, Rab4 selectively decreased the mature hERG protein expression on the plasma membrane. Mechanistically, Rab4 did not directly target the internalized hERG protein for recycling. Instead, Rab4 increased the expression level of the E3 ubiquitin ligase Nedd4-2 (Neural Precursor Cell-expressed Developmentally Downregulated Protein 4-2), which has been shown to mediate hERG ubiquitination and degradation (Guo et al., 2012). Nedd4-2 binding site mutations ∆1073 (binding site is removed) and Y1078A (binding site is modified) in hERG completely abolished the effect of Rab4. It has been shown that Nedd4-2 undergoes self-ubiquitination after targeting substrates (Bruce et al., 2008). My data further demonstrated that Rab4 decreased the degradation rate of Nedd4-2 and increased the rate of recycling. The increased Nedd4-2 then decreases hERG expression at the plasma membrane by targeting the PY-motif in the C-terminus of hERG channels. In summary, the present study showed that Rab4 decreases the expression and function of hERG potassium channels on the plasma membrane through enhancing the recycling of the ubiquitin ligase Nedd4-2. / Thesis (Master, Physiology) -- Queen's University, 2013-08-09 12:11:27.938

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Respiratory distress syndrome and pulmonary oedema often result in poor health and in the worst case scenario, death. Several studies have proposed that the eventual resolution of these dangerous conditions is due to active sodium reabsorption through the epithelial sodium channel (ENaC), which is crucial for lung fluid clearance. Although clinical prognosis can be improved by using glucocorticoid hormones to augment the ENaC-dependent removal of liquid from the lungs, we still require a better understanding of the underlying mechanism in order to improve treatments in the future. This thesis, therefore explores the role of serum / glucocorticoid-inducible protein kinase 1 (SGK1) and protein kinase A (PKA) in the responses of hormone-stimulated H441 human airway cells. Dexamethasone, a synthetic glucocorticoid hormone, is thought to evoke expression of the gene encoding SGK1 and, to become catalytically active, this gene product must then be phosphorylated via TORC2 and PDK1, protein kinases activated via the P13-kinase pathway. Once activated, SGK1 appears to exert control over the surface abundance of ENaC subunits by phosphorylation, and thus inactivating, a ubiquitin ligase (Nedd4-2), that normally mediate the withdrawal of ENaC subunits from the plasma membrane. Protein kinase A (PKA) may contribute to this control mechanism by also phosphorylating Nedd4-2. In order to clarify the way in which these pathways contribute to glucocorticoid-induced lung liquid clearance, the present thesis has explored the effects of dexamethasone and / or PKA activation upon the overall / surface expression of ENaC subunits, the activities of SGK1 and PKA and the phosphorylation status of physiologically-important residues within Nedd4-2 itself.