The role and mechanism of chaperones Calnexin/Calreticulin in which ALLN selectively rescues the trafficking defective of HERG-A561V mutation

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.

Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG‑A561V protein

“…At present, no study has described how to influence the trafficking of mutant HERG proteins by regulating key molecular chaperones in the CNX/CRT cycle. Our previous study verified that the two chaperone proteins CNX and CRT arrest the export of mutant HERG from the ER and empower it to refold into the correct native conformation and thus serve a role in preventing trafficking and degrading defective HERG mutant protein (19). The present study further evaluated the roles of CNX, CRT and ERP57 in trafficking the defective HERG-A561V mutant protein.…”

“…Compared with WT HERG, represented on a western blot by a 155-kDa band of mature HERG channel protein, the mutant HERG protein, represented on a western blot by a 135-kDa immature HERG protein band, is prone to misfolding and is retained in the ER ( 25 ). The mutant channel protein will combine with the WT protein to form a hybrid channel, producing two protein bands of 155 and 135 kDa on a western blot, and stay in the ER, thereby reducing the expression of the WT protein in the cell membrane ( 19 ). These mutant proteins, especially heterozygous channels, are not completely non-functional ( 5 ).…”

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

“…While we concluded that the latter mutants are aberrantly trafficked we could not rule out the fact that additional disease-causing mechanisms such as reduced stability or gating phenotypes (particularly for S217L 29 ) existed. However our data demonstrated that trafficking deficient Piezo1 mutants were differentially processed in heterologous expression systems as seen in the extensive studies of Kv11.1 18,49,[56][57][58][59] and CFTR 20,60,61 .…”

“…Again, using parallels with the Kv11.1 and CFTR literature, we attempted to rescue aberrant trafficking using two methods. The first was low temperature treatment, and the second was a pharmacological approach 18,22,49,50 . Specifically, a cohort of Kv11.1 and CFTR variants could be rescued at low temperature (<30 C) which is thought to improve protein folding and hence trafficking.…”

Loss-of-Function Piezo1 Mutations Display Altered Stability Driven by Ubiquitination and Proteasomal Degradation

“…Neither strategy was successful (Li et al, 2020). In the current study given the robust effects of ALLN on mutant Piezo1 protein levels we next asked the question of whether proteasome inhibition could rescue trafficking of Piezo1 mutants as shown for other ion channels (Wang et al, 2018).…”