scholarly journals Flavonoid regulation of EAG1 channels

2013 ◽  
Vol 141 (3) ◽  
pp. 347-358 ◽  
Author(s):  
Anne E. Carlson ◽  
Tinatin I. Brelidze ◽  
William N. Zagotta
Keyword(s):  
Cyclic Nucleotide ◽  
Neuronal Excitability ◽  
Physiological Role ◽  
Resonance Energy ◽  
Structural Similarity ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Cyclic Nucleotide Binding ◽  
Amino Terminal ◽  
The Brain

The voltage-gated, K+-selective ether á go-go 1 (EAG1) channel is expressed throughout the brain where it is thought to regulate neuronal excitability. Besides its normal physiological role in the brain, EAG1 is abnormally expressed in several cancer cell types and promotes tumor progression. Like all other channels in the KCNH family, EAG1 channels have a large intracellular carboxy-terminal region that shares structural similarity with cyclic nucleotide–binding homology domains (CNBHDs). EAG1 channels, however, are not regulated by the direct binding of cyclic nucleotides and have no known endogenous ligands. In a screen of biological metabolites, we have now identified four flavonoids as potentiators of EAG1 channels: fisetin, quercetin, luteolin, and kaempferol. These four flavonoids shifted the voltage dependence of activation toward more hyperpolarizing potentials and slowed channel deactivation. All four flavonoids regulated channel gating with half-maximal concentrations of 2–8 µM. The potentiation of gating did not require the amino-terminal or post-CNBHD regions of EAG1 channels. However, in fluorescence resonance energy transfer and anisotropy-based binding assays, flavonoids bound to the purified CNBHD of EAG1 channels. The CNBHD of KCNH channels contains an intrinsic ligand, a conserved stretch of residues that occupy the cyclic nucleotide–binding pocket. Mutations of the intrinsic ligand in EAG1 (Y699A) potentiated gating similar to flavonoids, and flavonoids did not further potentiate EAG1-Y699A channels. Furthermore, the Y699A mutant CNBHD bound to flavonoids with higher affinity than wild-type CNBHD. These results suggest that the flavonoids identified here potentiated EAG1 channels by binding to the CNBHD, possibly by displacing their intrinsic ligand. EAG1 channels should be considered as a possible target for the physiological effects of flavonoids.

scholarly journals Discovery and Characterization of a Distinct Cyclic Nucleotide Binding Pocket in HCN Channels

2014 ◽  
Vol 106 (2) ◽  
pp. 627a
Author(s):  
Anna Moroni ◽  
Marco Lolicato ◽  
Annalisa Bucchi ◽  
Cristina Arrigoni ◽  
Stefano Zucca ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Hcn Channels ◽  
Cyclic Nucleotide Binding

scholarly journals Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

2013 ◽  
Vol 142 (4) ◽  
pp. 351-366 ◽  
Author(s):  
Elena C. Gianulis ◽  
Qiangni Liu ◽  
Matthew C. Trudeau
Keyword(s):  
Cyclic Nucleotide ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Specific Interaction ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Nucleotide Binding ◽  
Cyclic Nucleotide Binding ◽  
Partial Restoration ◽  
Herg Channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.

scholarly journals The intrinsically liganded cyclic nucleotide–binding homology domain promotes KCNH channel activation

2017 ◽  
Vol 149 (2) ◽  
pp. 249-260 ◽  
Author(s):  
Yaxian Zhao ◽  
Marcel P. Goldschen-Ohm ◽  
João H. Morais-Cabral ◽  
Baron Chanda ◽  
Gail A. Robertson
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Voltage Sensor ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Channel Activation ◽  
Cyclic Nucleotide Binding ◽  
Slow Channel ◽  
Multiple Conformations

Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide–binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an “intrinsic ligand.” The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance–voltage (G–V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole–Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577–708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole–Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.

scholarly journals Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain

2016 ◽  
Vol 291 (34) ◽  
pp. 17907-17918 ◽  
Author(s):  
Eva Lörinczi ◽  
Matthew Helliwell ◽  
Alina Finch ◽  
Phillip J. Stansfeld ◽  
Noel W. Davies ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Cyclic Nucleotide Binding

scholarly journals Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

2021 ◽  
Author(s):  
Christopher Pfleger ◽  
Jana Kusch ◽  
Mahesh Kondapuram ◽  
Tina Schwabe ◽  
Christian Sattler ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

scholarly journals Structural and Energetic Analysis of Activation by a Cyclic Nucleotide Binding Domain

2008 ◽  
Vol 381 (3) ◽  
pp. 655-669 ◽  
Author(s):  
Stephen L. Altieri ◽  
Gina M. Clayton ◽  
William R. Silverman ◽  
Adrian O. Olivares ◽  
Enrique M. De La Cruz ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Energetic Analysis ◽  
Cyclic Nucleotide Binding Domain
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