scholarly journals The N-linker region of hERG1a upregulates hERG1b potassium channels

2021 ◽  
Author(s):  
Ashley Johnson ◽  
Taylor L Crawford ◽  
Matthew C Trudeau
Keyword(s):  
Plasma Membrane ◽  
Potassium Channels ◽  
Long Qt Syndrome ◽  
Action Potentials ◽  
Membrane Surface ◽  
Physiological Role ◽  
Pas Domain ◽  
Long Qt ◽  
Linker Region ◽  
Qt Syndrome

A major physiological role of hERG1 (human Ether-a-go-go-Related Gene) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the native cardiac IKr current in vivo. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, Long QT Syndrome and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal PAS (Per-Arnt-Sim) domain-N-linker region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labelled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the 1b domain did not have a measurable increase in current or biotinylated protein when co-expressed with hERG1a PAS domain-N-linker regions indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a PAS domain-N-linker region and the hERG1b N-terminal 1b domain. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216-220 within the hERG1a N-linker region that were necessary for the upregulation of hERG1b. Taken together, we propose that direct structural interactions between the hERG1a N-linker region and the hERG1b N-terminal 1b domain increase hERG1b at the plasma membrane. Mechanisms that enhance hERG1b current would be anticipated to shorten action potentials, which could be anti-arrhythmic, and may point toward hERG1b or the hERG1a N-linker as molecular targets for therapy for Long QT syndrome.

scholarly journals Long QT Syndrome-associated Mutations in the Per-Arnt-Sim (PAS) Domain of HERG Potassium Channels Accelerate Channel Deactivation

1999 ◽  
Vol 274 (15) ◽  
pp. 10113-10118 ◽  
Author(s):  
Jun Chen ◽  
Anrou Zou ◽  
Igor Splawski ◽  
Mark T. Keating ◽  
Michael C. Sanguinetti
Keyword(s):  
Potassium Channels ◽  
Long Qt Syndrome ◽  
Pas Domain ◽  
Long Qt ◽  
Qt Syndrome ◽  
Accelerate Channel

Abstract 15754: Genetically Engineered Stem Cell-Derived Model for Therapeutic Discovery in Long QT Syndrome

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jordan S Leyton-Mange ◽  
Min-Young Jang ◽  
Stacey N Lynch ◽  
Robert W Mills ◽  
Xaio Ling ◽  
...  
Keyword(s):  
Stem Cell ◽  
Long Qt Syndrome ◽  
Action Potentials ◽  
Biological Activities ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Hesc Line ◽  
Long Qt ◽  
Structure Activity ◽  
Qt Syndrome

Introduction: Long QT syndrome (LQTS) is caused by delayed ventricular repolarization and is a cause of sudden death. In a recent chemical screen with an in vivo zebrafish model of LQTS-type 2, we identified 2-methoxy-N-[4-methylphenyl] benzamide (2MMB), which shortens the ventricular action potential (Figure A). Methods: We initially used a zebrafish LQT2 model to perform a structure-activity relationship (SAR) study with 50 substituted benzanilide derivatives. We generated a human LQT-type 1 embryonic stem cell (hESC) model with a bi-allelic disruption of KCNQ1 in the hESC H7 line using CRISPR. KCNQ1-/- hESC-derived cardiomyocytes (hESC-CM) were electrophysiologically characterized and used to perform secondary structure activity studies for a subset of benzanilide derivatives (Figure A). Results: We observed a range of biological activities among the 50 analogs tested in the zebrafish assay, including several compounds with no activity and several that were more potent than 2MMB. Genetically engineered LQT1 hESC-CMs had diminished IKs current (Figure B) and prolonged action potentials that significantly shortened upon 2MMB treatment (Figure C). SAR studies in the LQT1 hESC-CMs confirmed improved potency for two of the compounds and lack of activity for one. A single discrepancy existed for a compound without activity in our zebrafish assay but significantly shortened action potentials in hESC-CMs. Conclusions: We report the use of a physiologically faithful genetically engineered LQT1 hESC line for therapeutic discovery in LQTS. Inclusion of hESC-CMs in future studies may complement animal testing by enabling direct screening on disease specific human tissue.

scholarly journals A Novel Mutation (T65P) in the PAS Domain of the Human Potassium Channel HERG Results in the Long QT Syndrome by Trafficking Deficiency

2002 ◽  
Vol 277 (50) ◽  
pp. 48610-48616 ◽  
Author(s):  
Aimée Paulussen ◽  
Adam Raes ◽  
Gert Matthijs ◽  
Dirk J. Snyders ◽  
Nadine Cohen ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Long Qt Syndrome ◽  
Novel Mutation ◽  
Pas Domain ◽  
Long Qt ◽  
Qt Syndrome

Long QT Syndrome 2 PAS Domain Variant Induces hERG1a/1b Subunit Imbalance in Patient-specific iPSC-cardiomyocytes

2021 ◽  
Author(s):  
Li Feng ◽  
Jianhua Zhang ◽  
ChangHwan Lee ◽  
Gina Kim ◽  
Fang Liu ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Protein Trafficking ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Pas Domain ◽  
Long Qt ◽  
Pathogenic Variants ◽  
Human Ipscs ◽  
Qt Syndrome ◽  
Domain Variant

Background - Inherited long QT syndrome type 2 (LQT2) results from variants in the KCNH2 gene encoding the hERG1 potassium channel. Two main isoforms, hERG1a and hERG1b, assemble to form tetrameric channel. The N-terminal Per-Arnt-Sim (PAS) domain, present only on hERG1a subunits, is a hotspot for pathogenic variants, but it is unknown whether PAS domain variants impact hERG1b expression to contribute to the LQT2 phenotype. We aimed to use patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the pathogenesis of the hERG1a PAS domain variant hERG1-H70R. Methods - Human iPSCs were derived from a LQT2 patient carrying the PAS domain variant hERG1-H70R. CRISPR/Cas9 gene editing produced isogenic control iPSC lines. Differentiated iPSC-CMs were evaluated for their electrophysiology, hERG1a/1b mRNA expression, and hERG1a/1b protein expression. Results - Action potentials from single hERG1-H70R iPSC-CMs were prolonged relative to controls, and voltage clamp studies showed an underlying decrease in I Kr with accelerated deactivation. In hERG1-H70R iPSC-CMs, transcription of hERG1a and hERG1b mRNA was unchanged compared to controls based on nascent nuclear transcript analysis, but hERG1b mRNA was significantly increased as was the ratio of hERG1b/hERG1a in mRNA complexes, suggesting post-transcriptional changes. Expression of complex glycosylated hERG1a in hERG1-H70R iPSC-CMs was reduced due to impaired protein trafficking, whereas the expression of the complex glycosylated form of hERG1b was unchanged. Conclusions - Patient-specific hERG1-H70R iPSC-CMs reveal a newly appreciated mechanism of pathogenesis of the LQT2 phenotype due to both impaired trafficking of hERG1a and maintained expression of hERG1b that produces subunit imbalance and reduced I Kr with accelerated deactivation.

Altered re-excitation thresholds and conduction of extrasystolic action potentials contribute to arrhythmogenicity in murine models of long QT syndrome

2012 ◽  
Vol 206 (3) ◽  
pp. 164-177 ◽  
Author(s):  
R. M. Duehmke ◽  
S. Pearcey ◽  
J. D. Stefaniak ◽  
L. Guzadhur ◽  
K. Jeevaratnam ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Action Potentials ◽  
Murine Models ◽  
Long Qt ◽  
Qt Syndrome

scholarly journals Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits.

1999 ◽  
Vol 274 (35) ◽  
pp. 25188
Author(s):  
Laura Franqueza ◽  
Monica Lin ◽  
Jiaxiang Shen ◽  
Mark T. Keating ◽  
Michael C. Sanguinetti
Keyword(s):  
Potassium Channels ◽  
Long Qt Syndrome ◽  
Long Qt ◽  
Qt Syndrome

Abstract 17137: Unmasking Pathological Mechanisms of Long QT Syndrome KCNH2 H70R Variant Using Human iPSC-Cardiomyocytes

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Li Feng ◽  
Gina Kim ◽  
Catherine A Eichel ◽  
Fang Liu ◽  
Evi Lim ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Herg Channel ◽  
Patient Specific ◽  
Delayed Rectifier ◽  
Pas Domain ◽  
Loss Of Function ◽  
Long Qt ◽  
Unrelated Control ◽  
Qt Syndrome ◽  
Human Ipsc

Introduction: Inherited long QT syndrome type 2 (LQT2) results from loss-of-function mutations in the KCNH2 gene encoding the hERG channel, which conducts I Kr , the rapid component of the delayed rectifier K + current. The N-terminal Per-Arnt-Sim (PAS) domain present on the hERG1a subunit, but not the hERG1b subunit, is the site of multiple LQT2-linked missense variants. The mechanism of loss of function by many of these missense PAS variants is unclear given conflicting results from different heterologous expression systems expressing hERG1a. Hypothesis: Patient-specific LQT2 human iPSC-cardiomyocytes (hiPSC-CMs) which naturally express hERG1a/1b carrying the KCNH2 H70R variant in the PAS domain will exhibit loss of I Kr associated with APD prolongation due to impaired channel protein trafficking. Methods and Results: Human iPSCs were derived from a patient carrying the LQT2-associated PAS domain mutation KCNH2 H70R, which has been reported to cause impaired hERG channel trafficking without effects on channel gating when expressed in HEK 293 cells but accelerated deactivation kinetics of I hERG when expressed in Xenopus laevis oocytes. Two clones of KCNH2 H70R and unrelated control hiPSCs (DF19-9-11T) were differentiated using monolayer-base, small molecule protocol to CMs, evaluated with whole-cell patch clamp. Action potentials from single hiPSC-CMs paced at 1Hz were prolonged in the hERG-H70R group compared to control (APD 90 439.9 ± 15.3 ms vs. 363.7 ± 29.0ms, p =0.003, H70R: n=11, control: n=9, Temp 36 ± 1°C). Voltage clamp studies showed hERG-H70R hiPSC-CMs had a significantly smaller peak tail I Kr current density (1.1 ± 0.3 vs. 2.9 ± 0.5 pA/pF, p <0.001, H70R: n=11, control: n=7, Temp 36 ± 1°C). The voltage dependence of I Kr activation (V½ and k) were not affected by the mutation; however, the fast (τf) and slow (τs) deactivation time constants were significantly decreased in hERG-H70R hiPSC-CMs. Further, Western blot characterization revealed impaired trafficking of hERG-H70R channels relative to control. Conclusions: The LQT2 PAS domain variant hERG-H70R results in loss of function of I Kr by both reduced membrane trafficking and accelerated deactivation of hERG in a hiPSC-CMs model which informs therapeutic approaches.

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