Novel characteristics of a trafficking-defective G572R-hERG channel linked to hereditary long QT syndrome

Lian, Jing; Huang, Na; Zhou, Jianhua; Shi-jun, GE; Huang, Xiaoyan; Huo, Jianhua; Liu, Liying; Xu, Wei-Qun; Zhang, Shun; Yang, Xi; Zhou, Jian; Huang, Chen

doi:10.1016/s0828-282x(10)70439-6

Cited by 7 publications

(8 citation statements)

References 33 publications

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“…We found that untagged hERG channels and EGFP/hERG channel were expressed both on the cell surface and within the cytoplasm, which indicated that EGFP/hERG fusion protein did not impact hERG channels targeting the cell surface. Researchers of the R863X, G601S, G604S, G572R and T65P mutations study all used EGFP/hERG to study protein trafficking defect, which indicated that EGFP/hERG can be used to study the subcellular localization of hERG and hERG mutation (Furutani et al, 1999; Paulussen et al, 2002; Teng et al, 2004; Huo et al, 2008; Lian et al, 2010). However, Claassen et al (2008) recently studied that the extent of labelling with anti‐hERG antibodies of HEK293 cells expressing EGFP/hERG channels was less compared with that expressing hERG/EGFP channels, which suggests that EGFP/hERG channels might have a protein trafficking defect.…”

Section: Discussionmentioning

confidence: 99%

“…EGFP tagged to either the C‐terminus (hERG/EGFP) or N‐terminus (EGFP/hERG) of hERG channels is used to study defective protein trafficking for several years (Furutani et al, 1999; Petrecca et al, 1999; Teng et al, 2004; Rossenbacker et al, 2005). In our former researches about G604S—hERG and G572R—hERG mutants associate with LQT2 (Huo et al, 2008; Lian et al, 2010), we used EGFP/hERG to study protein trafficking and ion channel subcellular localization. However, GFP has some limitations as a reporter gene.…”

Section: Introductionmentioning

confidence: 99%

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The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

et al. 2011

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EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

et al. 2011

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“…G572R-hERG and E637K-hERG are two mutant forms of the hERG channel that have been previously reported as trafficking-deficient [20], [21]. However, the exact process by which they are retained in the ER is not well understood.…”

Section: Introductionmentioning

confidence: 99%

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

et al. 2012

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BackgroundLong QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.MethodspcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.ConclusionOur results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.

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“…Cel‐miR‐67 was taken as random negative controls. The hERG currents were elicited by voltage clamp protocol as previously described . Cells were depolarized to test voltage between –60 and +60 mV in –10 mV increments for 3 seconds from a holding potential of –80 mV, followed by –40 mV for 4 seconds to elicit tail currents (Fig.…”

Section: Resultsmentioning

confidence: 99%

miRNAs Regulate hERG

Lian

Guo

Huang

et al. 2016

Cardiovasc electrophysiol

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Novel characteristics of a trafficking-defective G572R-hERG channel linked to hereditary long QT syndrome

Cited by 7 publications

References 33 publications

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

miRNAs Regulate hERG

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