Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

Marques-Carvalho, Maria J.; Morais-Cabral, J.H.

doi:10.1107/s1744309112004216

Cited by 4 publications

(4 citation statements)

References 18 publications

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“…Abrogation of interactions may release KCNQ1 from a state of functional downregulation, thus contributing to the increase in I Ks current density following adrenergic stimulation. However, further studies are needed to delineate the contribution of PKAmediated effects on hERG-KCNQ1 interactions from direct cAMP-binding, particularly as recent reports of the crystal structures of COOH-terminal domains in other EAG-family channels contend that cAMP does not directly bind to the CNBD (6,20). It is also possible that both pathways are involved in the mechanisms regulating repolarization reserve.…”

Section: Discussionmentioning

confidence: 99%

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP

Darling

Vernon

Giovanniello

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.

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Section: Discussionmentioning

confidence: 99%

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP

Darling

Vernon

Giovanniello

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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“…Protein was purified by histidine-tag affinity and size-exclusion chromatography, as previously described. 28 The fusion MBP-BD-C1 was expressed and purified with the same basic procedure except 250 mM KCl was used instead of NaCl to minimize aggregation. Protein for crystallization trials was dialyzed overnight at 4°C against gel-filtration buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, and 5 mM DTT) in the presence of thrombin.…”

Section: Methodsmentioning

confidence: 99%

“…28 A data set was collected at the ID14-4 beamline of the European Synchrotron Radiation Facility (ESRF), and the structure was solved by molecular replacement. Model refinement was done in PHENIX 29 ; TLS refinement was applied.…”

Section: Crystallization Data Collection and Refinementmentioning

confidence: 99%

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

Marques-Carvalho

Sahoo

Muskett

et al. 2012

Journal of Molecular Biology

104

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“…The CNBHD is structurally similar to that of the CNG and HCN channels, but unlike those channels, the hERG CNBHD only weakly associates with cyclic nucleotides and does not undergo regulation by cyclic nucleotides[ 2 ]. In place of a cyclic nucleotide, the CNBHD in the hERG family of channels is bound by an intrinsic ligand, which is primarily composed of a three amino acid piece of the CNBHD itself[ 3 – 6 ].…”

Section: Introductionmentioning

confidence: 99%

Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Liu

Trudeau

2015

PLoS ONE

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Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal ‘eag’ domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

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Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

Cited by 4 publications

References 18 publications

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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