HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms

Akil Puckerin, Kelly A. Aromolaran, Donald D. Chang, R. Suzanne Zukin, Henry M. Colecraft, Mohamed Boutjdir, Ademuyiwa S. Aromolaran

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Background: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. Objective: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Methods: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. Results: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Conclusion: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

Original languageEnglish (US)
JournalHeart Rhythm
DOIs
StateAccepted/In press - 2016

Fingerprint

Fluorescence Resonance Energy Transfer
Immunoprecipitation
Mutation
Long QT Syndrome
HEK293 Cells
Electrophysiology
Potassium Channels
Cardiac Myocytes
Ether
Cardiac Arrhythmias
Guinea Pigs
Phenotype
Genes
Therapeutics

Keywords

  • Cardiomyocytes
  • Guinea pig
  • HEK293
  • HERG 1a
  • HERG 1b
  • I
  • KCNH2
  • LQT2
  • Potassium channel
  • Truncation mutants

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

Puckerin, A., Aromolaran, K. A., Chang, D. D., Zukin, R. S., Colecraft, H. M., Boutjdir, M., & Aromolaran, A. S. (Accepted/In press). HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms. Heart Rhythm. https://doi.org/10.1016/j.hrthm.2016.01.012

HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms. / Puckerin, Akil; Aromolaran, Kelly A.; Chang, Donald D.; Zukin, R. Suzanne; Colecraft, Henry M.; Boutjdir, Mohamed; Aromolaran, Ademuyiwa S.

In: Heart Rhythm, 2016.

Research output: Contribution to journalArticle

Puckerin, Akil ; Aromolaran, Kelly A. ; Chang, Donald D. ; Zukin, R. Suzanne ; Colecraft, Henry M. ; Boutjdir, Mohamed ; Aromolaran, Ademuyiwa S. / HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms. In: Heart Rhythm. 2016.
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abstract = "Background: The human ether-{\`a}-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. Objective: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Methods: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. Results: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Conclusion: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.",
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T1 - HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms

AU - Puckerin, Akil

AU - Aromolaran, Kelly A.

AU - Chang, Donald D.

AU - Zukin, R. Suzanne

AU - Colecraft, Henry M.

AU - Boutjdir, Mohamed

AU - Aromolaran, Ademuyiwa S.

PY - 2016

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N2 - Background: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. Objective: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Methods: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. Results: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Conclusion: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

AB - Background: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. Objective: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Methods: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. Results: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Conclusion: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

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KW - HERG 1b

KW - I

KW - KCNH2

KW - LQT2

KW - Potassium channel

KW - Truncation mutants

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