Abstract
Long QT (LQT) type 2 (LQT2) is caused by HERG mutation. L539fs/47 encodes a truncated protein, and its mechanisms in HERG mutation are unknown. HERG mutation plasmids were overexpressed in HEK293T cells, respectively, followed by analyzing lysates with Western blot. Transfected HEK293T cells were treated with or without N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) and Propranolol (Prop) at 24 or 48 h. HERG-WT, HERG-A561V, WT/A561V, HERG-L539fs/47, WT/L539fs/47, and Calnexin (CNX)/Calreticulin (CRT) protein expression and their interactions were detected by Western blot and immunoprecipitation. Each group with HERG currents (Ikr) were detected by Patch-clamp technique. Treated with ALLN, the expression of mature HERG protein and the CNX/CRT protein increased. The interaction of HERG-A561V and WT/A561V protein with the chaperone CNX/CRT increased significantly. The maximum peak currents and tail currents density increased by 70% and 73%, respectively, while maximal peak currents density (24%) and tail currents density (19%) were slight increased in WT-HERG cells. Treated with Prop, the expression and interaction of mature HERG and chaperones CNX/CRT had no difference in each group. The maximal currents and tail currents density were slight increased. CNX/CRT might play a crucial role in the trafficking-deficient process and degradation of HERG-A561V mutant protein, however they had no effect on L539fs/47 HERG due to protein transport deletion. ALLN can restore HERG-A561V mutant protein trafficking process and rescue the dominant negative suppression of WT-HERG.
Highlights
The congenital long QT (LQT) syndrome is a heterogeneous genetic disease characterized by delayed cardiac repolarization, prolonged electrocardiographic QT intervals, the development of ventricular arrhythmias (Torsades de pointes) and sudden death, often in young healthy individuals, children and teenagers [1,2,3]
To determine whether ALLN corrects the translocation of HERG-A561V mutant protein by inhibiting proteasome degradation pathway, the expression of HERG-WT, HERG-A561V, HERG-L539fs/47, HERG-WT/A561V, HERG-WT/L539fs/47 and CNX/CRT protein were detected by Western blot in transfected HEK293T cells treated with or without ALLN (10 μmol/l) after 24 or 48 h
The protein intensity of CNX and CRT were increased significantly in HERG-L539fs/47 cells treated with 10 μmol/L ALLN, while herg and Activating transcription factor 6 (ATF6) were not (Figure 1C/c). These results suggest that ALLN can correct the transport barrier of A561V mutant proteins, and the molecular chaperones may promote the degradation of HERG-A561V mutant proteins, but not L539fs/47
Summary
The congenital long QT (LQT) syndrome is a heterogeneous genetic disease characterized by delayed cardiac repolarization, prolonged electrocardiographic QT intervals, the development of ventricular arrhythmias (Torsades de pointes) and sudden death, often in young healthy individuals, children and teenagers [1,2,3]. LQT type 2 (LQT2) is the second most common type, caused by mutations in KCNH2 or the human ether-a-go-go-related gene (hERG) [4,5]. Defective trafficking of mutant channels to the cell membrane represent the most dominant mechanism of hERG channel dysfunction in LQT2 [6]. More than 500 hERG mutations having been identified, amongst which majority caused LQT2 due to HERG protein trafficking deficiency [7]. The endoplasmic reticulum (ER) is an important system for protein synthesis and cell processing, which is the strict quality control system to assemble protein correctly through the Golgi apparatus and reach the final site [8,9]. The protein is misfolded and trapped in the ER and the disorder of transport occurs
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