Regulation of the Human Ether-à-Go-Go-Related Gene (hERG) Channel by RAB4 Through Neural Precursor Cell-Expressed Developmentally Downregulated Protein 4-2 (NEDD4-2)
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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.