Novel Mechanism Associated With an Inherited Cardiac Arrhythmia

Furutani, Michiko; Trudeau, Matthew C.; Hagiwara, Nobuhisa; Seki, Akiko; Gong, Qiuming; Zhou, Zhengfeng; Imamura, Shin Ichiro; Nagashima, Hirotaka; Kasanuki, Hiroshi; Takao, Atsuyoshi; Momma, Kazuo; January, Craig T.; Robertson, Gail A.; Matsuoka, Rumiko

doi:10.1161/01.cir.99.17.2290

Cited by 173 publications

(95 citation statements)

References 26 publications

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“…Western blot analyses of WT hERG overexpression in several systems, including HEK-293 cells, have shown two protein bands at 135 kDa (immature) and 155 kDa (mature) that represent posttranslational core and complex glycosylation of hERG protein, respectively (2,15,19,45). We next transfected the missense mutations, G601S and N470D hERG (12,16,36), that form trafficking-deficient channel proteins when studied in noncardiac mammalian systems (3,16,45). Western blot analysis in the cardiomyocytes showed that G601S and N470D hERG generate 135-kDa protein bands but only weak or no 155-kDa protein bands, indicating that for control conditions these two mutations generate immature protein that then does not undergo normal Golgi processing to the mature protein.…”

Section: Resultsmentioning

confidence: 99%

“…WT hERG with appropriate nucleotide changes (⌬TAC2342-2344) resulting in ⌬Y475 hERG was generated using site-directed mutagenesis and verified by DNA sequencing. G601S and N470D hERG were generated as previously described (16,45). hERG cDNAs for neonatal mouse cardiomyocyte experiments were generated as endotoxin free using the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

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Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes

Lin

Holzem

Anson

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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Mutations in humanether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming ␣-subunits that coassemble to form rapidly activating delayed rectifier K ϩ current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The ⌬Y475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5-to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes

Lin

Holzem

Anson

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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“…To directly address whether TRC8 selects misfolded populations of hERG for degradation, we studied the LQT2 pore mutation G601S and the CNBD mutation F805C, both of which cause misfolding and ERAD (3)(4)(5)(6). Growth at 27°C allows some folding and trafficking of hERG-G601S, albeit inefficiently compared with WT (5).…”

Section: Trc8 Promotes Degradation Of Misfolded Mutant Herg-g601s-mentioning

confidence: 99%

“…The hERG polypeptide contains N-and C-terminal cytosolic domains around a central transmembrane region that tetramerizes to form the channel pore, implicating cytosolic and transmembrane factors in quality control. LQT2 mutations that disrupt folding are found throughout the hERG sequence, for example G601S in the pore region and F805C in the C-terminal cyclic nucleotide binding domain (CNBD) (3)(4)(5)(6). The chaperone Hsp90 is necessary for hERG trafficking (7).…”

mentioning

confidence: 99%

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

Hantouche

Williamson

Valinsky

et al. 2017

Journal of Biological Chemistry

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Edited by George N. DeMartinoCardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG.

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“…Some familial forms of LQT syndrome are caused by the production of mutant hERG proteins that are inappropriately retained in the ER (26)(27)(28). Remarkably, this deficit can sometimes be overcome by treatment with certain hERGbinding drugs that somehow promote the subsequent processing and transport of the mutant channels to the plasma membrane, where they function normally (26,29,30).…”

Section: Discussionmentioning

confidence: 99%

A Drosophila behavioral mutant, down and out ( dao ), is defective in an essential regulator of Erg potassium channels

Fergestad

Sale

Bostwick

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out ( dao ). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels.

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Novel Mechanism Associated With an Inherited Cardiac Arrhythmia

Cited by 173 publications

References 26 publications

Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes

Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

A Drosophila behavioral mutant, down and out ( dao ), is defective in an essential regulator of Erg potassium channels

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Novel Mechanism Associated With an Inherited Cardiac Arrhythmia

Cited by 173 publications

References 26 publications

Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

A Drosophila behavioral mutant, down and out ( dao ), is defective in an essential regulator of Erg potassium channels

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Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes

Properties of WT and mutant hERG K⁺ channels expressed in neonatal mouse cardiomyocytes