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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.
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???displayArticle.pmcLink???PMC3581696 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Dendrogram and basic architecture of channels of the cyclic nucleotideâregulated superfamily. (A) Phylogenetic tree depicting the evolutionary relationships of the KCNH, CNG, and HCN ion channel families, computed with Cobalt (Papadopoulos and Agarwala, 2007) and displayed with Dendroscope (Huson et al., 2007). (B) Schematic of the architecture of a single mEAG1 subunit, with the structure of the CNBHD shown in magenta (Marques-Carvalho et al., 2012) and the tyrosine and leucine of the intrinsic ligand shown in blue. Inset includes a close-up view of the tyrosine and leucine of the intrinsic ligand superimposed with cAMP (yellow) from the HCN2 structure (Zagotta et al., 2003). Superposition of the mEAG1 and mHCN2 structures was done through residues in the β roll.
Figure 2. Chemical structures of screened flavonoids and structurally related compounds. The following flavonoids were included in this screen: fisetin (2-(3,4-dihydroxyphenyl)-3,7-dihydroxychromen-4-one), kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one), luteolin (2-(3,4-dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone), quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one), acacetin (5,7-dihydroxy-2-(4-methoxyphenyl)chromen-4-one), apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), catechin ((2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol), myricetin (3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), galangin (3,5,7-trihydroxy-2-phenylchromen-4-one), and narnigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one). The steroid hormone 17-β-estradiol ((17β)-estra-1,3,5(10)-triene-3,17-diol) and the cyclic nucleotides cAMP (3â²5â²-cyclic adenosine monophosphate) and cGMP (3â²5â²-cyclic guanosine monophosphate) were also included in this screen. Box highlights the compounds that potentiated mEAG1 currents.
Figure 3. Fisetin modulated mEAG1 currents. (A) Representative current traces of mEAG1 channels recoded in the inside-out patch configuration in the absence (black), presence (red), and after washout (gray) of 30 µM fisetin. (B) The averaged conductanceâvoltage relationship of mEAG1 channels with 0 (black) or 30 µM fisetin (red) fit with a Boltzmann function (n = 6). (C) The tail current recorded at â120 mV after a voltage step to 50 mV in the absence (black) and presence (red) of 30 µM fisetin. Fits of these tail currents with single exponentials reported the time constant of deactivation: 5.3 ms before and 13.9 ms with 30 µM fisetin. (D) Plot of the ÎV1/2 versus free fisetin concentration fit with a Hill equation (n = 5). The half-maximal concentration of fisetin was 3.5 ± 0.4 µM. (E) Plot of the time constant of mEAG1 deactivation versus free fisetin concentration, fit with a Hill equation (n = 5). The half-maximal concentration of fisetin was 6.6 ± 3.7 µM.
Figure 4. Fisetin potentiated mEAG1 currents recorded in both inside-out and outside-out patches. Box plots depicting the distributions of the shifts of the V1/2 of the conductanceâvoltage relationships in the presence of 10 µM fisetin applied to mEAG1 channels and recorded in the inside-out or outside-out configuration of the patch-clamp technique (n = 4â7).
Figure 5. Fisetin regulated mEAG1 channels lacking their amino-terminal or post-CNBHD regions. (A) Representative current traces from mEAG1 Î2â173 channels, recorded in the inside-out configuration of the patch-clamp technique, in the absence (black) and presence (red) of 30 µM fisetin. Patches were held at â100 mV, and currents were evoked by 3-s voltage steps to depolarizing potentials ranging from â100 to +100 mV, in 20-mV increments, and returned to â100 mV for 1 s. (B) Representative conductanceâvoltage relationships of mEAG1 Î2â173 channels with 0 (black) or 30 µM fisetin (red) fit with the product of two Boltzmann functions. For both curves, the V1/2i, si, and sa were held constant at â4.5, 5.9, and 7 mV, respectively. For 0 fisetin, the V1/2a was equal to â19.8 mV, and for 30 µM fisetin, the V1/2a was â39.4 mV. (C) Tail currents at â100 mV after a step to â40 mV were fit with single exponentials (green lines) to give time constants of 116 ms without and 239 ms with 30 µM fisetin. (D) Representative current traces of mEAG1 Î821â989 recorded in the absence (black) and presence (red) of 30 µM fisetin. Patches were held at â100 mV, and currents were evoked by a series of 100-ms voltage steps ranging from â140 to +50 mV, in 10-mV increments, followed by a 100-ms voltage pulse to â120 mV. (E) The averaged conductanceâvoltage relationship of mEAG1 channels lacking their post-CNBHD region, EAG1 Î821â989, recorded in the inside-out patch configuration in the absence (black) and presence (red) of 30 µM fisetin (n = 4). The data were fit with Boltzmann functions to give a V1/2 of â31.9 mV without and â50.6 mV with 30 µM fisetin. (F) Tail currents at â120 mV after a step to +50 mV fit with single exponentials (green lines) to give time constants of 5.2 ms without and 13.4 ms with 30 µM fisetin.
Figure 6. Absorption spectra for fisetin and quercetin overlap with the emission spectrum of tryptophan. Shown are the absorption spectra of 10 µM fisetin (blue) and quercetin (black) (left axis). Also shown is the emission spectrum of 4 µM of free tryptophan (red) excited with 295 nm (right axis).
Figure 7. Quercetin and fisetin bound to purified CNBHD of mEAG1 channels. (A and B) The inner filterâcorrected and background-subtracted emission spectra of 4 µM of purified C-linker/CNBHD (A) and 4 µM of free tryptophan (B) recorded with 0 or 100 µM fisetin as indicated. (C and D) The inner filterâcorrected and background-subtracted tryptophan fluorescence emission spectra of 4 µM of purified C-linker/CNBHD (C) or 4 µM of free tryptophan (D) recorded with various concentrations of quercetin, as indicated. (E) Plot of the change in the peak emission fluorescence intensity (at 341 nm) of the C-linker/CNBHD versus total quercetin concentration. These data were fit with Eq. 6 to report an apparent binding affinity of 93 ± 69 µM. (F) Fluorescence anisotropy of fisetin plotted versus the total concentration of wild-type or Y699A mutant CNBHD. The data were fit with Eq. 7 to yield Kdâs of 111 µM for wild-type and 30 µM for the Y699A mutant.
Figure 8. Fisetin did not potentiate mEAG1 channels with a mutated intrinsic ligand. (A) Alignment of the intrinsic ligand from each of the eight members of the KCNH channel family. Line on the top marks the boundaries of the intrinsic ligand, and the arrow on the bottom denotes the Y699 residue in mEAG1 channels. (B) The conductanceâvoltage relationship of wild-type (black) and Y699A (green) mEAG1 channels recoded in the inside-out patch configuration (n = 17). (C) Representative current traces from Y699A mutant channels recorded in the absence (black) and presence (red) of 30 µM fisetin. (D) Box plot distribution of the V1/2 of the conductanceâvoltage relationship for both wild-type and mEAG1 Y699A channels with 0 or 30 µM fisetin as indicated (n = 6â10). A two-way ANOVA reported differences between these four groups of data (P < 0.01). The TukeyâKramer post-hoc test revealed significant differences between treatments, denoted with an asterisk. Treatments with nonsignificant differences are also indicated (n.s.). (E) Box plot distribution of the time constant of deactivation for both wild-type and mEAG1 Y699A channels with 0 or 30 µM fisetin as indicated (n = 6â10). A two-way ANOVA reported differences between these four groups of data (P < 0.05). The TukeyâKramer post-hoc test also revealed significant and nonsignificant differences between treatments, indicated with an asterisk or n.s., respectively.
Figure 9. Flavone and tyrosine structures. (A) Chemical structure of the flavone subclass of flavonoids and (B) structure of the amino acid tyrosine.
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