Pethő Z et al. (2024), A synthetic flavonoid derivate in the plasma me...

XB-ART-61034

FEBS J 2024 Jun 02;29111:2354-2371. doi: 10.1111/febs.17105.

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A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Pethő Z , Pajtás D , Piga M , Magyar Z , Zakany F , Kovacs T , Zidar N , Panyi G , Varga Z , Papp F .

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Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

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Species referenced: Xenopus laevis
Genes referenced: hvcn1

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	Fig. 1Effect of flavonoid compounds on the fluorescence signal of CiHv1-E241C. Representative fluorescence responses to a depolarization from −60 to +100 mV, recorded from Xenopus oocytes expressing CiHv1 channels labeled with TAMRA-MTS at the E241C position (red), and exposed to different treatments: Mix A (A), Oxophench (B) and Mix B (E). Fh is the baseline fluorescence at the holding potential, ΔFpeak is negative peak fluorescence change during the voltage step, ΔFss (steady state) is the change in fluorescence at the end of the voltage step, ΔFtail is the maximum change in fluorescence during repolarization after the voltage step and ΔFhook = ΔFtail − ΔFss; the relative fluorescence change: ΔF/Fh, simplified to ΔF/F. ΔFSS/Fh and ΔFhook/Fh are quantified in (C) and (D), respectively, and in (F). Representative fluorescence traces were selected from n oocytes, N different batches: n/N control = 8/3, n/N Mix A = 3/3, n/N Oxophench = 8/3, (D) n/N Mix B = 3/3. Data in (C), (D), and (F) represent mean ± SEM. Statistical comparison was performed using one-way ANOVA in (C), (D), and with Student's t-test in (F).
	Fig. 2Compound library of synthetic flavonoid derivates Mix A on the left side shows the structure of modified flavonoids, where a butylamino (BuNH2) group, an anisidine group, or a morpholine group were introduced on C-6 or C-7. Mix B on the right side shows amino acid-conjugated synthetic flavonoid molecules, where C-7 was functionalized with either one amino acid (Leu, Val, Met, Phe-O-Me) or with a phenylalanine-containing dipeptide (Phe-Leu, Phe-Phe, Phe-Trp). The purple, blue, and green colors represent the color coding shown in the other figures, i.e. purple is the color for Mix A, blue for Mix B and green for Oxophench.
	Fig. 3Oxophench acts on the CiHv1 channel in a time-, voltage- and dose-dependent manner. (A) TAMRA-MTS-labeled CiHv1 fluorescence responses to a voltage step from −60 to +100 mV at different concentrations of Oxophench: 0.01, 0.1, 1, and 10 μm. Darker colors indicate increasing concentrations. The dashed line represents the base line fluorescence value. (B) Representative fluorescence responses upon depolarization from −60 to +100 mV after applying continuous perfusion of 10 μm Oxophench. Time points are 0 s (red), 75 s (orange), 120 s (yellow), and 360 s (green). The dashed line represents the base line fluorescence value. (C) Voltage-dependence of VCF signal (ΔFtail/Fh = ΔF/Ftail) normalized to amplitude measured at +100 mV under control conditions (red) and 10 μm Oxophench (green). (D) Proton currents measured at depolarizations to +100 and +20 mV in the absence and presence of 10 μm Oxophench (red and green, respectively). The dashed line represents the base line value (proton current is zero). (E) Voltage-dependence of CiHv1 steady-state conductance (G) normalized to control conductance value measured +60 mV under control (red) and 10 μm Oxophench application (green). (F) Remaining Current Fraction (RCF) was calculated by dividing the peak current at the equilibrium block by the peak current in the absence of Oxophench. The solid line was fitted with a three-parameter inhibitor vs. response equation (see the details in the Materials and methods section). Representative fluorescence and current traces were selected from N = 3 different batches. Lines in (C), (E), and (F) are sigmoidal fits of the indicated data points. Data in (C), (D), and (F) represent mean ± SEM.
	Fig. 4Oxophench incorporates into the membrane of HEK293 cells and oocytes. (A) Representative confocal microscopy images of HEK293 cells stained with 10 μm Oxophench for 1 min compared to before staining (0 min). Scale bar = 10 μm. (B) Excitation (purple) and emission (green) spectra of Oxophench (C) TAMRA holding fluorescence (Fh) of stained, uninjected oocytes (not expressing CiHv1) normalized to the fluorescence value measured at the initial time point, before and after 10 μm Oxophench perfusion. (D) TAMRA holding fluorescence (Fh) of stained oocytes expressing CiHv1-E241C normalized to the fluorescence value measured at the initial time point. Substances applied are 100 μm phenylalanine (Phe), 100 μm flavone backbone (flavone) and 10 μm Oxophench. (E, F) Quantification and representative VCF signals of data represented in (C). (F) Effects of 10 μm Oxophench on TMA-DPH fluorescence anisotropy inversely related to membrane fluidity as determined using spectrofluorimetry. Representative fluorescence data points in (C) and (D) were selected from n = 3 measurements. The green dashed lines in (C) and (D) are exponential fits to the indicated data points. Data in (E) (n = 3) represent mean ± SEM, with statistical comparison performed using one-way ANOVA. Data in (G) represent mean ± SEM of n = 15 biological replicates from three independent experiments, with statistical comparison performed using Student's t-test.
	Fig. 5Oxophench quenches TAMRA fluorescence. (A) Representative caption of a molecular docking experiment with Oxophench (blue molecule, highlighted in green ellipse) docked into the extracellular POPC membrane bilayer leaflet (gray) in the vicinity of TAMRA-MTS (green molecule) bound to CiHv1 (red) at position E241C. This figure was generated by using ucsf chimera x and autodock (details are in the Materials and methods: Molecular modeling) (B) Representative spectrofluorimetric TAMRA-MTS fluorescence under control (red) or in the presence of Oxophench (green): 10 μm, 100 μm, 500 μm, and 1 mm. Darker colors indicate increasing concentrations. (C) Fluorescence intensity (F) relative to control (F0) as a function of wavelength at different Oxophench concentrations (F/F0 vs λ) (SEM is indicated by black lines, N = 3). (D) Fluorescence intensities normalized (Fnorm = F/F0) to control (dashed line) in the presence of 1 mm Oxophench (green), 15 mm tryptophan (Trp, gray), 15 and 50 mm histidine (His, yellow and orange), and 50 mm alanine (Ala, black). (E) Solid black lines represent common three-state kinetic model calculation of VCF signal generation under control (left) and 10 μm Oxophench (right) at different membrane potentials (from −60 mV up to +100 mV), compared to experimental traces (colored lines). Fluorescence Representative graphics, traces, and models in (A), (B), and (E), respectively, were selected from n = 3 measurements. Solid green line in (C) is an exponential fit to the indicated data points. Data in (C) and (D) (N = 3) represent mean ± SEM, with statistical comparison in (D) performed using one-way ANOVA.
	Fig. 6Oxophench affects VCF signal in quencher-mutant CiHv1-E241C constructs. (A–D) Representative fluorescence responses (A, C) and proton currents (B, D) to a voltage step from −60 to +100 mV, under control (red) and 10 μm Oxophench (green) from oocytes labeled with TAMRA-MTS, expressing Ci-HV1 E241C containing the following mutations: H179W/H188W (A, B), H179A (C, D). (E, F) Quantification of steady-state and hook ΔF/F under control (red) and 10 μm Oxophench (green). (G) Working model of Oxophench (purple X) on the VCF signal: the model assumes two major VSD (Voltage-Sensitive Domain; green) movements among three states traced by the attached TAMRA (orange star). The resting (1), the intermediate (2) and the activated (3) states are associated with different fluorescence intensities (symbolized by different size of orange star. Smaller size represents less fluorescence). TAMRA is in close proximity to the membrane, and thus Oxophench, in the resting state (1). This leads to TAMRA quenching since Oxophench has strong quenching ability. Upon depolarization, TAMRA first moves away from the lipid bilayer and the quencher Oxophench from state (1) to (2), leading to dequenching. Subsequently, there is a second motion from state 2 to 3, when the dye approaches the lipid bilayer and a quenching His or point-mutated Trp residue (white circle), but at the same time moves further away from Oxophench and thus fluorescence further increases. Representative fluorescence and current traces were selected from n oocytes/N different batches: H179W/H188W: n/N control = 5/3, n/N Oxophench = 5/3; H179A: n/N control = 4/3, n/N Mix A = 4/3. Data bars in (E) and (F) represent mean ± SEM, with statistical comparison performed using Student's t-test.
	Fig. 7Quality assessment of CiHv1-TAMRA model geometry. (A) Error values, based on the statistics of non-bonded atom-atom interactions in the reported structure, are plotted as a function of the amino acid position of a sliding 9-residue window. Regions of the structure that can be rejected at the 95% confidence level are yellow, i.e., 5% of a good protein structure is expected to have an error value above this level. Regions that can be rejected at the 99% level are shown in red. (B) Ramachandran plot of the model. Phi values on the x-axis and the Psi values on the y-axis to predict the possible conformation of the peptide. The angle spectrum in each axis is from −180° to +180°. The regions highlighted in dark and light blue show amino acids (black dots) in core and allowed conformations, respectively. The amino acids highlighted in red are outside the allowed conformation range.