4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene

Abstract

Background Long QT syndrome type 2 (LQT2) is caused by mutations in the KCNH2 /human ether-à-go-go-related gene (hERG). Some hERG genetic mutation-associated diseases are alleviated by hERG-specific drug chaperones (glycerol, dimethyl sulfoxide, trimethylamine N-oxide, thapsigargin), delayed rectifier K + current (IKr) blockers methanesulfonanilide E4031, the antihistamine astemizole, or the prokinetic drug cisapride, and the anti-arrhythmic drug quinidine. Meanwhile, many in vivo and in vitro studies have reported the efficacy of 4-phenylbutyric acid (4-PBA) in diseases with inherited genetic mutations. This study aims to explore potential therapeutic agents for hERG/G572R mutated ion channel. Methods pcDNA3/hERG [wild type (WT)]-FLAG and pcDNA3/hERG (G572R)-FLAG plasmids were transfected into HEK293 cells. A western blot (WB) experiment was conducted to analyze protein expression. Quantitative real-time polymerase chain reaction (qPCR) was used to analyze the messenger RNA (mRNA) expression levels in the WT/G572R heterozygous HEK293 cell model treated with or without 4-PBA. The interaction between WT/G572R and BIP (GRP78), GRP94, and 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation protein 1 (HRD1) was tested by co-immunoprecipitation (co-IP). To investigate the effect of 4-PBA on the WT/G572R channel current, we used electrophysiological assays (patch-clamp electrophysiological recordings). Results The results showed that WT/G572R activated the ATF6 pathway in the endoplasmic reticulum stress (ERS), the ERS response markers GRP78, GRP94, and calreticulin (CRT)/calnexin (CNX), and HRD1, which decreased after application of the ERS inhibitor 4-PBA. The results of co-IP confirmed that the ability of hERG interacted with GRP78, GRP94, and HRD1. Moreover, 4-PBA increased the current of WT/G572R and reversed the gating kinetics of the WT/G572R channel. Conclusions 4-PBA corrects hERG channel transport defects by inhibiting excessive ERS and the endoplasmic reticulum-associated degradation (ERAD)-related gene E3 ubiquitin ligase HRD1. Additionally, 4-PBA improved WT/G572R channel current. 4-PBA is expected to be developed as a new treatment method for LQT2.

Ion channel trafficking implications in heart failure

“…Indeed, a study described that 90% of hERG variants inhibited the trafficking from the ER to the Golgi ( 226 ). These variants cause hERG transport disorders by stimulating the UPR and leading the degradation of the ion channel protein ( 182 ). Variants on KCNQ1 were also revealed to play a role in the abnormal KCNQ1 trafficking, leading to HF and long QT syndrome ( 227 , 228 ).…”