The Role of Monoubiquitination in Endocytic Degradation of Human Ether-a-go-go-related Gene (hERG) Channels under Low K+ Conditions

Tao Sun,Jun Guo,Heidi Shallow,Tonghua Yang,Jianmin Xu,Wentao Li,Christian Hanson,James G Wu,Xian Li,Hamid Massaeli,Shetuan Zhang

doi:10.1074/jbc.m110.198408

Abstract

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.

Highlights

The human ether-a-go-go-related gene2 encodes the pore-forming subunits of the rapidly activating delayed rectifier Kϩ channels (IKr) [1, 2]
Monoubiquitination Is Involved in Low Kϩ-induced Internalization and Degradation of wild type (WT) human ether-a-go-go-related gene (hERG) Channels but Not S624T Mutant hERG Channels—As shown in Fig. 1A, the hERG proteins extracted from hERG-human embryonic kidney (HEK) cells cultured in normal MEM (5 mM Kϩ) displayed two bands with molecular masses of 155 and 135 kDa, representing the mature fully glycosylated form in the plasma membrane (155 kDa) and the immature core-glycosylated form residing in the endoplasmic reticulum (135 kDa) [26, 27]
Our work showed that Ub is involved in endocytic degradation of cell surface hERG channels under low Kϩ conditions [6, 7], the nature of Ub-hERG interactions is unknown

Summary

Introduction

The human ether-a-go-go-related gene (hERG)2 encodes the pore-forming subunits of the rapidly activating delayed rectifier Kϩ channels (IKr) [1, 2]. Monoubiquitination Is Involved in Low Kϩ-induced Internalization and Degradation of WT hERG Channels but Not S624T Mutant hERG Channels—the hERG proteins extracted from hERG-HEK cells cultured in normal MEM (5 mM Kϩ) displayed two bands with molecular masses of 155 and 135 kDa, representing the mature fully glycosylated form in the plasma membrane (155 kDa) and the immature core-glycosylated form residing in the endoplasmic reticulum (135 kDa) [26, 27].

Results

Conclusion