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

Wen Tang; Dihui Cai; Yin Fu; Zequn Zheng; Xiaoyan Huang; Rami N Khouzam; Yongfei Song; Jiangfang Lian

doi:10.21037/jtd-23-1252

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

J Thorac Dis. 2023 Aug 31;15(8):4472-4485. doi: 10.21037/jtd-23-1252. Epub 2023 Aug 28.

Authors

Wen Tang^#¹, Dihui Cai^#¹, Yin Fu¹, Zequn Zheng¹, Xiaoyan Huang², Rami N Khouzam³, Yongfei Song², Jiangfang Lian^{1

2}

Affiliations

¹ Department of Cardiology, The Affiliated Lihuili Hospital, Ningbo University, Ningbo, China.
² Ningbo Institute of Innovation for Combined Medicine and Engineering, Ningbo, China.
³ Division of Cardiovascular Diseases, Department of Internal Medicine, University of Tennessee Health Science Center, Memphis, TN, USA.

^# Contributed equally.

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.

Keywords: 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation protein 1 (HRD1); Endoplasmic reticulum stress (ERS); endoplasmic reticulum-associated degradation (ERAD); ubiquitin-proteasome pathway (UPP); unfolded protein response (UPR).