The potassium channel protein KCNH2 is encoded by KCNH2 gene, and there are more than 300 mutations of KCNH2. Unfolded protein response (UPR) is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER). The present study aimed to explore the UPR process and the role of activating transcription factor 6 (ATF6) in the abnormal expression of potassium voltage-gated channel subfamily H member 2 (KCNH2)A561V. The wild-type (wt) KCNH2 and A561V mutant KCNH2 was constructed with his-tag. The 293 cells were used and divided into KCNH2wt+KCNH2A561V, KCNH2wt and KCNH2A561V groups. The expression levels of ATF6 and KCNH2 in different groups were detected by Western blotting, reverse transcription-quantitative PCR, immunofluorescence and immuno-coprecipitation assays. The protein types and abundance of immuno-coprecipitation samples were analyzed by mass spectrometry. The proteomic analysis of the mass spectrometry results was carried out by using the reactome database and GO (Gene Ontology) tool. The mRNA expression levels of KCNH2 and ATF6 in the KCNH2wt+KCNH2A561V group were higher compared with the KCNH2A561V group. However, the full-length protein expression of ATF6 was inhibited, indicating that ATF6 was highly activated and a substantial number of ATF6 was sheared in KCNH2wt+KCNH2A561V group compared with control group. Furthermore, A561V-KCNH2 mutation leading to the accumulation of the immature form of KCNH2 (135 kDa bands) in ER, resulting in the reduction of the ratio of 155 kDa/135 kDa. In addition, the abundance of UPR-related proteins in the KCNH2A561V group was higher compared with the KCNH2wt+KCNH2A561V group. The 'cysteine biosynthetic activity' of GO:0019344 process and the 'positive regulation of cytoplasmic translation activity' of GO:2000767 process in the KCNH2A561V group were higher compared with the KCNH2wt+KCNH2A561V group. Hence, co-expression of wild-type and A561V mutant KCNH2 in 293 cells activated the UPR process, which led to the inhibition of protein translation and synthesis, in turn inhibiting the expression of KCNH2. These results provided a theoretical basis for clinical treatment of Long QT syndrome.

Emergency Medical Center Ningbo Yinzhou No 2 Hospital Ningbo Zhejiang China; Department of General Surgery Ningbo No 2 Hospital Ningbo China Department of Cardiology Ningbo Medical Center LiHuiLi Hospital Ningbo China

Zobrazit více v PubMed

Shah SR, Park K, Alweis R. Long QT Syndrome: A Comprehensive Review of the Literature and Current Evidence. Curr Probl Cardiol. 2019;44:92–106. doi: 10.1016/j.cpcardiol.2018.04.002. PubMed DOI

Wallace E, Howard L, Liu M, O’Brien T, Ward D, Shen S, Prendiville T. Long QT Syndrome: Genetics and Future Perspective. Pediatr Cardiol. 2019;40:1419–1430. doi: 10.1007/s00246-019-02151-x. PubMed DOI PMC

Neira V, Enriquez A, Simpson C, Baranchuk A. Update on long QT syndrome. J Cardiovasc Electrophysiol. 2019;30:3068–3078. doi: 10.1111/jce.14227. PubMed DOI

Restivo M, Caref EB, Kozhevnikov DO, El-Sherif N. Spatial dispersion of repolarization is a key factor in the arrhythmogenicity of long QT syndrome. J Cardiovasc Electrophysiol. 2004;15:323–331. doi: 10.1046/j.1540-8167.2004.03493.x. PubMed DOI

Kapplinger JD, Giudicessi JR, Ye D, Tester DJ, Callis TE, Valdivia CR, Makielski JC, et al. Enhanced Classification of Brugada Syndrome-Associated and Long-QT Syndrome-Associated Genetic Variants in the SCN5A-Encoded Na(v)1.5 Cardiac Sodium Channel. Circ Cardiovasc Genet. 2015;8:582–595. doi: 10.1161/CIRCGENETICS.114.000831. PubMed DOI PMC

Li G, Shi R, Wu J, Han W, Zhang A, Cheng G, Xue X, Sun C. Association of the hERG mutation with long-QT syndrome type 2, syncope and epilepsy. Mol Med Rep. 2016;13:2467–2475. doi: 10.3892/mmr.2016.4859. PubMed DOI PMC

Vandenberg JI, Perry MD, Perrin MJ, Mann SA, Ke Y, Hill AP. hERG K(+) channels: structure, function, and clinical significance. Physiol Rev. 2012;92:1393–1478. doi: 10.1152/physrev.00036.2011. PubMed DOI

Niven JE, Vahasoyrinki M, Kauranen M, Hardie RC, Juusola M, Weckstrom M. The contribution of Shaker K+ channels to the information capacity of Drosophila photoreceptors. Nature. 2003;421:630–634. doi: 10.1038/nature01384. PubMed DOI

Shimizu W. The long QT syndrome: therapeutic implications of a genetic diagnosis. Cardiovasc Res. 2005;67:347–356. doi: 10.1016/j.cardiores.2005.03.020. PubMed DOI

Wang Y, Shen T, Fang P, Zhou J, Lou K, Cen Z, Qian H, et al. The role and mechanism of chaperones Calnexin/Calreticulin in which ALLN selectively rescues the trafficking defective of HERG-A561V mutation. Biosci Rep. 2018;38:BSR20171269. doi: 10.1042/BSR20171269. PubMed DOI PMC

Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995;80:795–803. doi: 10.1016/0092-8674(95)90358-5. PubMed DOI

Kagan A, Yu Z, Fishman GI, McDonald TV. The dominant negative LQT2 mutation A561V reduces wild-type HERG expression. J Biol Chem. 2000;275:11241–11248. doi: 10.1074/jbc.275.15.11241. PubMed DOI

Tester DJ, Will ML, Haglund CM, Ackerman MJ. Compendium of cardiac channel mutations in 541 consecutive unrelated patients referred for long QT syndrome genetic testing. Heart Rhythm. 2005;2:507–517. doi: 10.1016/j.hrthm.2005.01.020. PubMed DOI

Ma S, Zhao Y, Cao M, Sun C. Human etheragogorelated gene mutation L539fs/47hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway. Int J Mol Med. 2019;43:1253–1262. doi: 10.3892/ijmm.2019.4049. PubMed DOI PMC

Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2008;7:1013–1030. doi: 10.1038/nrd2755. PubMed DOI

Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000;2:326–332. doi: 10.1038/35014014. PubMed DOI

Yuan ZL, Zhang ZX, Mo YZ, Li DL, Xie L, Chen MH. Inhibition of extracellular signal-regulated kinase downregulates endoplasmic reticulum stress-induced apoptosis and decreases brain injury in a cardiac arrest rat model. Physiol Res. 71:413–423. doi: 10.33549/physiolres.934882. PubMed DOI PMC

Estebanez B, de Paz JA, Cuevas MJ, Gonzalez-Gallego J. Endoplasmic Reticulum Unfolded Protein Response, Aging and Exercise: An Update. Front Physiol. 2018;9:1744. doi: 10.3389/fphys.2018.01744. PubMed DOI PMC

Senft D, Ronai ZA. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci. 2015;40:141–148. doi: 10.1016/j.tibs.2015.01.002. PubMed DOI PMC

Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K. Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6alpha and 6beta that activates the mammalian unfolded protein response. Mol Cell Biol. 2001;21:1239–1248. doi: 10.1128/MCB.21.4.1239-1248.2001. PubMed DOI PMC

Glembotski CC, Rosarda JD, Wiseman RL. Proteostasis and Beyond: ATF6 in Ischemic Disease. Trends Mol Med. 2019;25:538–550. doi: 10.1016/j.molmed.2019.03.005. PubMed DOI PMC

Glembotski CC. Roles for ATF6 and the sarco/endoplasmic reticulum protein quality control system in the heart. J Mol Cell Cardiol. 2014;71:11–15. doi: 10.1016/j.yjmcc.2013.09.018. PubMed DOI PMC

Ryoo HD. Long and short (timeframe) of endoplasmic reticulum stress-induced cell death. FEBS J. 2016;283:3718–3722. doi: 10.1111/febs.13755. PubMed DOI PMC

Bahar E, Kim JY, Yoon H. Quercetin attenuates manganese-induced neuroinflammation by alleviating oxidative stress through regulation of apoptosis, iNOS/NF-kappaB and HO-1/Nrf2 pathways. Int J Mol Sci. 2017;18:1989. doi: 10.3390/ijms18091989. PubMed DOI PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. PubMed DOI

Gardner BM, Pincus D, Gotthardt K, Gallagher CM, Walter P. Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb Perspect Biol. 2013;5:a013169. doi: 10.1038/nrneph.2017.129. PubMed DOI PMC

Gong Q, Anderson CL, January CT, Zhou Z. Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol. 2002;283:H77–H84. doi: 10.1152/ajpheart.00008.2002. PubMed DOI

Zhang KP, Yang BF, Li BX. Translational toxicology and rescue strategies of the hERG channel dysfunction: biochemical and molecular mechanistic aspects. Acta Pharmacol Sin. 2014;35:1473–1484. doi: 10.1038/aps.2014.101. PubMed DOI PMC

Foo B, Williamson B, Young JC, Lukacs G, Shrier A. hERG quality control and the long QT syndrome. J Physiol. 2016;594:2469–2481. doi: 10.1113/JP270531. PubMed DOI PMC

Wu Y, Huang X, Zheng Z, Yang X, Ba Y, Lian J. Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG-A561V protein. Int J Mol Med. 2021;48:159. doi: 10.3892/ijmm.2021.4992. PubMed DOI PMC

Hong M, Luo S, Baumeister P, Huang JM, Gogia RK, Li M, Lee AS. Underglycosylation of ATF6 as a novel sensing mechanism for activation of the unfolded protein response. J Biol Chem. 2004;279:11354–11363. doi: 10.1074/jbc.M309804200. PubMed DOI

Shen J, Chen X, Hendershot L, Prywes R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev Cell. 2002;3:99–111. doi: 10.1016/S1534-5807(02)00203-4. PubMed DOI

Shen J, Prywes R. ER stress signaling by regulated proteolysis of ATF6. Methods. 2005;35:382–389. doi: 10.1016/j.ymeth.2004.10.011. PubMed DOI

Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS, Goldstein JL. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell. 2000;6:1355–1364. doi: 10.1016/S1097-2765(00)00133-7. PubMed DOI

Liu M, Liu H, Parthiban P, Kang GJ, Shi G, Feng F, Zhou A, et al. Inhibition of the unfolded protein response reduces arrhythmia risk after myocardial infarction. J Clin Invest. 2021;131:e147836. doi: 10.1172/JCI147836. PubMed DOI PMC

van Dijk EL, Schilders G, Pruijn GJ. Human cell growth requires a functional cytoplasmic exosome, which is involved in various mRNA decay pathways. RNA. 2007;13:1027–1035. doi: 10.1261/rna.575107. PubMed DOI PMC

Rozpedek W, Pytel D, Mucha B, Leszczynska H, Diehl JA, Majsterek I. The role of the PERK/eIF2alpha/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress. Curr Mol Med. 2016;16:533–544. doi: 10.2174/1566524016666160523143937. PubMed DOI PMC

Teske BF, Wek SA, Bunpo P, Cundiff JK, McClintick JN, Anthony TG, Wek RC. The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress. Mol Biol Cell. 2011;22:4390–4405. doi: 10.1091/mbc.e11-06-0510. PubMed DOI PMC