I
            <sub>Kr</sub>
            drug response is modulated by
            <i>KCR1</i>
            in transfected cardiac and noncardiac cell lines

Kupershmidt, Sabina; Yang, Iris Chun-Hui; Hayashi, Kenshi; Wei, Jian; Chanthaphaychith, Siprachanh; Petersen, Christina I.; Johns, David C.; George, Alfred L.; Roden, Dan M.; Balser, Jeffrey R.

doi:10.1096/fj.02-1057fje

Cited by 52 publications

(55 citation statements)

References 41 publications

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“…However, T24D1.4, the closest worm homologue to KCR1, which is a protein originally isolated in a screen to identify a noninactivating K ϩ current from rat cerebellum (9), did modify the activity of unc-103 (n500) in vivo. Although the function of KCR1 is not fully defined, the protein appears to be expressed in human heart and influences HERG sensitivity to drug blockage in heterologous expression systems and transfected cardiac myocytes (10). F37C12.12 (mec-14) has the closest amino acid similarity to Drosophila Hyperkinetic, and electrophysiological studies of cultured Drosophila giant neurons demonstrate that mutations in Hyperkinetic result in increased excitability and altered sensitivity to classical K ϩ channel blockers (17).…”

Section: Discussionmentioning

confidence: 99%

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

Petersen

McFarland

Stepanovic

et al. 2004

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

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Human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of IKr, a cardiac K ؉ channel. Although many commonly used drugs block IKr, in certain individuals, this action evokes a paradoxical life-threatening cardiac rhythm disturbance, known as the acquired long QT syndrome (aLQTS). Although aLQTS has become the leading cause of drug withdrawal by the U.S. Food and Drug Administration, DNA sequencing in aLQTS patients has revealed HERG mutations only in rare cases, suggesting that unknown HERG modulators are often responsible. By using the worm Caenorhabditis elegans, we have developed in vivo behavioral assays that identify candidate modulators of unc-103, the worm HERG orthologue. By using RNA-interference methods, we have shown that worm homologues of two HERG-interacting proteins, Hyperkinetic and K channel regulator 1 (KCR1), modify unc-103 function. Examination of the human KCR1 sequence in patients with drug-induced cardiac repolarization defects revealed a sequence variation (the substitution of isoleucine 447 by valine, I447V) that occurs at a reduced frequency (1.1%) relative to a matched control population (7.0%), suggesting that I447V may be an allele for reduced aLQTS susceptibility. This clinical result is supported by in vitro studies of HERG dofetilide sensitivity by using coexpression of HERG with wild-type and I447V KCR1 cDNAs. Our studies demonstrate the feasibility of using C. elegans to assay and potentially identify aLQTS candidate genes.C aenorhabditis elegans unc-103 shares 70% amino acid identity with HERG in the conserved transmembrane and pore regions of the protein (see Scheme 1).Studies using unc-103 promoter GFP reporter constructs reveal expression of unc-103 in body-wall muscle, egg-laying muscles, pharyngeal muscles, and neurons that innervate these tissues (D.J.R, J.H.T., and R. Garcia, unpublished data). Worm strains carrying mutations in this gene, unc-103 (n500) and unc-103 (e1597), were isolated in screens for locomotion-defective mutants (1). Analysis of both of these neomorphic mutant strains revealed the same mutation: conversion of a conserved alanine in the S6 transmembrane domain to a threonine (A334T, indicated in bold above). Our studies reveal that these mutant worms exhibit profound neuromuscular defects, and the severity of these defects is sensitive to modulators that decrease the level of mutant channel activity. Further, the human homologues of these modulators may have physiologically relevant interactions with human ether-a-go-go-related gene (HERG) and represent acquired long QT syndrome (aLQTS) candidate genes. MethodsMolecular Biology. The human K channel regulator 1 (KCR1) cDNA, an IMAGE clone (no. 650823) was purchased from Research Genetics (Huntsville, AL). Site-directed mutagenesis was performed as described (2). For in vitro cRNA transcription, we used the SP6 mMessage mMachine high-yield capped RNA transcription kit (Ambion, Austin, TX). For RNA interference (RNAi) vectors, we PCR-amplified fragments of 0.9-1.5 kb of the target gene f...

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Section: Discussionmentioning

confidence: 99%

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

Petersen

McFarland

Stepanovic

et al. 2004

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

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“…The HERG(N598Q) mutation was introduced into the same backbone using the Stratagene QuikChange site-directed mutagenesis kit. The human KCR1 cDNA was subcloned into the pCGI vector for bicistronic expression with enhanced green fluorescent protein (GFP) (16). ALG10 (kindly provided by Dr. Markus Aebi, ETH Zurich, Zurich, Switzerland) was inserted into the same vector for mammalian cell expression.…”

Section: Methodsmentioning

confidence: 99%

“…The same conditions were used for experiments performed with the HERG(N598Q) mutant. The cells were cultured as described (16). Cells exhibiting green fluorescence were chosen for current recordings.…”

Section: Methodsmentioning

confidence: 99%

“…Cells exhibiting green fluorescence were chosen for current recordings. Membrane currents were recorded essentially as described (16). Data acquisition was carried out using an Axopatch 200B amplifier and pCLAMP8.0 software (Axon Instruments, Foster City, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The K ϩ channel regulator KCR1 (38) protects heterologously expressed HERG current (I HERG ) from block by the antiarrhythmic drugs dofetilide, quinidine, and sotalol over clinically relevant concentration ranges (16). Moreover, a polymorphism associated with a reduced risk for aLQTS was shown to enhance KCR1-induced protection of I HERG from drug block in vitro (17).…”

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HERG Is Protected from Pharmacological Block by α-1,2-Glucosyltransferase Function

Nakajima¹,

Hayashi²,

Viswanathan

et al. 2007

Journal of Biological Chemistry

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The HERG (human ether-à-go-go-related gene) protein, which underlies the cardiac repolarizing current I Kr , is the unintended target for many pharmaceutical agents. Inadvertent block of I Kr , known as the acquired long QT syndrome (aLQTS), is a leading cause for drug withdrawal by the United States Food and Drug Administration. Hence, an improved understanding of the regulatory factors that protect most individuals from aLQTS is essential for advancing clinical therapeutics in broad areas, from cancer chemotherapy to antipsychotics and antidepressants. Here, we show that the K ؉ channel regulatory protein KCR1, which markedly reduces I Kr drug sensitivity, protects HERG through glucosyltransferase function. KCR1 and the yeast ␣-1,2-glucosyltransferase ALG10 exhibit sequence homology, and like KCR1, ALG10 diminished HERG block by dofetilide. Inhibition of cellular glycosylation pathways with tunicamycin abrogated the effects of KCR1, as did expression in Lec1 cells (deficient in glycosylation). Moreover, KCR1 complemented the growth defect of an alg10-deficient yeast strain and enhanced glycosylation of an Alg10 substrate in yeast. HERG itself is not the target for KCR1-mediated glycosylation because the dofetilide response of glycosylation-deficient HERG(N598Q) was still modulated by KCR1. Nonetheless, our data indicate that the ␣-1,2-glucosyltransferase function is a key component of the molecular pathway whereby KCR1 diminishes I Kr drug response. Incorporation of in vitro data into a computational model indicated that KCR1 expression is protective against arrhythmias. These findings reveal a potential new avenue for targeted prevention of aLQTS.I Kr , an important component of the cardiac delayed rectifier K ϩ current, is required for repolarization of the human cardiac action potential. Mutations in HERG (human ether-à-go-gorelated gene), which encodes the ␣-subunit of the channel complex, are responsible for the chromosome 7-linked form (LQT2) of the congenital long QT syndrome (LQTS), 2 an arrhythmia syndrome characterized by action potential prolongation and delayed cardiac repolarization. LQTS causes the atypical polymorphic ventricular tachycardia torsade de pointes, leading to sudden cardiac death (1-3). A far more commonly acquired form of LQTS (aLQTS) is precipitated by exposure to a wide range of therapeutic compounds that block HERG. aLQTS represents a significant problem for public health and the pharmaceutical industry 3 and is typically observed in predisposed patients receiving treatment for noncardiac illnesses. Indeed, about half of all drug withdrawals since 1998 were in response to potential pro-arrhythmic effects linked to aLQTS (5-7).Numerous drug families bind the HERG channel and suppress I Kr in vitro, yet, surprisingly, few patients experience aLQTS. Clinically silent ion channel gene mutations have been characterized in families with cases of aLQTS, indicating that even the drug-induced form of the disorder may carry a genetic component (8 -15). However, mutations in the gene that ...

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QT prolongation through hERG K⁺ channel blockade: Current knowledge and strategies for the early prediction during drug development

Recanatini

Poluzzi

Masetti

et al. 2004

Medicinal Research Reviews

257

116

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Prolongation of the QT interval of the electrocardiogram is a typical effect of Class III antiarrhythmic drugs, achieved through blockade of potassium channels. In the past decade, evidence has accrued that several classes of drugs used for non-cardiovascular indications may prolong the QT interval with the same mechanism (namely, human ether-a-go-go-related gene (hERG) K(+) channel blockade). The great interest in QT prolongation is because of several reasons. First, drug-induced QT prolongation increases the likelihood of a polymorphous ventricular arrhythmia (namely, torsades de pointes, TdP), which may cause syncope and degenerate into ventricular fibrillation and sudden death. Second, the fact that several classes of drugs, such as antihistamines, fluoroquinolones, macrolides, and neuroleptics may cause the long QT syndrome (LQTS) raises the question whether this is a class effect (e.g., shared by all agents of a given pharmacological class) or a specific effect of single agents within a class. There is now consensus that, in most cases, only a few agents within a therapeutic class share the ability to significantly affect hERG K(+) channels. These compounds should be identified as early as possible during drug development. Third, QT prolongation and interaction with hERG K(+) channels have become surrogate markers of cardiotoxicity and have received increasing regulatory attention. This review briefly outlines the mechanisms leading to QT prolongation and the different strategies that can be followed to predict this unwanted effect. In particular, it will focus on the approaches recently proposed for the in silico screening of new compounds.

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I _Kr drug response is modulated by KCR1 in transfected cardiac and noncardiac cell lines

Cited by 52 publications

References 41 publications

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

HERG Is Protected from Pharmacological Block by α-1,2-Glucosyltransferase Function

QT prolongation through hERG K⁺ channel blockade: Current knowledge and strategies for the early prediction during drug development

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I Kr drug response is modulated by KCR1 in transfected cardiac and noncardiac cell lines

Cited by 52 publications

References 41 publications

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

HERG Is Protected from Pharmacological Block by α-1,2-Glucosyltransferase Function

QT prolongation through hERG K+ channel blockade: Current knowledge and strategies for the early prediction during drug development

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I _Kr drug response is modulated by KCR1 in transfected cardiac and noncardiac cell lines

QT prolongation through hERG K⁺ channel blockade: Current knowledge and strategies for the early prediction during drug development