Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance

Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.

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hERG1a N-terminal eag domain–containing polypeptides regulate homomeric hERG1b and heteromeric hERG1a/hERG1b channels: A possible mechanism for long QT syndrome

The Journal of General Physiology ◽

10.1085/jgp.201110683 ◽

2011 ◽

Vol 138 (6) ◽

pp. 581-592 ◽

Cited By ~ 21

Author(s):

Matthew C. Trudeau ◽

Lisa M. Leung ◽

Elon Roti Roti ◽

Gail A. Robertson

Keyword(s):

Amino Acids ◽

Long Qt Syndrome ◽

Outward Current ◽

Inhibitory Effect ◽

Pas Domain ◽

Long Qt ◽

Current Voltage ◽

Qt Syndrome ◽

Herg Channels

Human ether-á-go-go–related gene (hERG) potassium channels are critical for cardiac action potential repolarization. Cardiac hERG channels comprise two primary isoforms: hERG1a, which has a regulatory N-terminal Per-Arnt-Sim (PAS) domain, and hERG1b, which does not. Isolated, PAS-containing hERG1a N-terminal regions (NTRs) directly regulate NTR-deleted hERG1a channels; however, it is unclear whether hERG1b isoforms contain sufficient machinery to support regulation by hERG1a NTRs. To test this, we constructed a series of PAS domain–containing hERG1a NTRs (encoding amino acids 1–181, 1–228, 1–319, and 1–365). The NTRs were also predicted to form from truncation mutations that were linked to type 2 long QT syndrome (LQTS), a cardiac arrhythmia disorder associated with mutations in the hERG gene. All of the hERG1a NTRs markedly regulated heteromeric hERG1a/hERG1b channels and homomeric hERG1b channels by decreasing the magnitude of the current–voltage relationship and slowing the kinetics of channel closing (deactivation). In contrast, NTRs did not measurably regulate hERG1a channels. A short NTR (encoding amino acids 1–135) composed primarily of the PAS domain was sufficient to regulate hERG1b. These results suggest that isolated hERG1a NTRs directly interact with hERG1b subunits. Our results demonstrate that deactivation is faster in hERG1a/hERG1b channels compared to hERG1a channels because of fewer PAS domains, not because of an inhibitory effect of the unique hERG1b NTR. A decrease in outward current density of hERG1a/hERG1b channels by hERG1a NTRs may be a mechanism for LQTS.

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Probucol aggravates long QT syndrome associated with a novel missense mutation M124T in the N-terminus of HERG

Clinical Science ◽

10.1042/cs20030351 ◽

2004 ◽

Vol 107 (2) ◽

pp. 175-182 ◽

Cited By ~ 33

Author(s):

Kenshi HAYASHI ◽

Masami SHIMIZU ◽

Hidekazu INO ◽

Masato YAMAGUCHI ◽

Hidenobu TERAI ◽

...

Keyword(s):

Missense Mutation ◽

Long Qt Syndrome ◽

Xenopus Oocytes ◽

Torsade De Pointes ◽

Qt Prolongation ◽

Delayed Rectifier ◽

Long Qt ◽

N Terminus ◽

Qt Syndrome ◽

Herg Channels

Patients with LQTS (long QT syndrome) with a mutation in a cardiac ion channel gene, leading to mild-to-moderate channel dysfunction, may manifest marked QT prolongation or torsade de pointes only upon an additional stressor. A 59-year-old woman had marked QT prolongation and repeated torsade de pointes 3 months after initiation of probucol, a cholesterol-lowering drug. We identified a single base substitution in the HERG gene by genetic analysis. This novel missense mutation is predicted to cause an amino acid substitution of Met124→Thr (M124T) in the N-terminus. Three other relatives with this mutation also had QT prolongation and one of them had a prolonged QT interval and torsade de pointes accompanied by syncope after taking probucol. We expressed wild-type HERG and HERG with M124T in Xenopus oocytes and characterized the electrophysiological properties of these HERG channels and the action of probucol on the channels. Injection of the M124T mutant cRNA into Xenopus oocytes resulted in expression of functional channels with markedly smaller amplitude. In both HERG channels, probucol decreased the amplitude of the HERG tail current, decelerated the rate of channel activation, accelerated the rate of channel deactivation and shifted the reversal potential to a more positive value. The electrophysiological study indicated that QT lengthening and cardiac arrhythmia in the two present patients were due to inhibition of IKr (rapidly activating delayed rectifier K+ current) by probucol, in addition to the significant suppression of HERG current in HERG channels with the M124T mutation.

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Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.4.h1748 ◽

2000 ◽

Vol 279 (4) ◽

pp. H1748-H1756 ◽

Cited By ~ 62

Author(s):

Eckhard Ficker ◽

Dierk Thomas ◽

Prakash C. Viswanathan ◽

Adrienne T. Dennis ◽

Silvia G. Priori ◽

...

Keyword(s):

Plasma Membrane ◽

Long Qt Syndrome ◽

Qt Interval ◽

Mammalian Cells ◽

Treatment Modalities ◽

Delayed Rectifier ◽

Long Qt ◽

Qt Syndrome ◽

Incubation Temperatures ◽

Herg Channels

Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG ( KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K+ currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.

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