Proteostatic regulation of hERG1 isoforms and LQT2 mutants by MKRN1

Loss-of-function mutations in type 1 human ether-à-go-go-related gene (hERG1) potassium channels are associated with the ventricular arrhythmia type 2 long QT syndrome (LQT2). Most LQT2-causing missense mutations lead to impaired membrane trafficking and enhanced endoplasmic reticulum (ER)-associated degradation of hERG1. Multiple types of E3 ubiquitin ligases differentially mediate the degradation of misfolded mutant hERG1 proteins at the ER and plasma membrane, the molecular details of which remain poorly understood. Herein, we aim to elucidate the molecular mechanisms by which the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1) regulates the protein quality control of hERG1. Biochemical and immunofluorescence analyses were employed to study the effect of MKRN1 on hERG1 proteostasis in HEK293T cells, mouse HL-1 atrial cells, and mouse and rat heart tissues. Electrophysiological investigations were applied to examine the functional expression of hERG1. Molecular modeling was used to explore the interaction between MKRN1 and hERG1. We demonstrated that MKRN1 was a novel binding partner of two hERG1 isoforms (hERG1a, hERG1b), probably by interacting with the carboxyl-terminal region of both isoforms, as well as the amino-terminal region of hERG1a. MKRN1 downregulated total hERG1 protein level and potassium current density, mainly by enhancing the degradation of ER-localized core-glycosylated immature hERG1 through the ubiquitin-proteasome system. MKRN1 promoted the ER-associated degradation of the misfolded LQT2-causing hERG1 mutants A561V and G601S. Suppression of endogenous MKRN1 activity by RNA interference partially corrected the trafficking defect of the G601S mutant. Treatment with 1 µM ebastine, a clinically used antihistamine and recently demonstrated MKRN1 inhibitor, also rescued the forward trafficking of A561V and G601S, as well as increasing the potassium current density of G601S. 250 nM ebastine was sufficient to achieve near-maximal rescue of hERG1-G601S protein maturation and functional current. Ebastine produced little or no additional G601S rescue effect under MKRN1 knockdown conditions. These findings are consistent with the notion that MKRN1 regulates ER proteostasis of immature hERG1 proteins in cardiomyocytes, and plays an essential role in controlling the membrane trafficking of LQT2-causing hERG1 mutants. Our study highlights the therapeutic potential of ebastine in LQT2 patients.