Liu QN and Trudeau MC (2015), Eag Domains Regulate LQT Mutant hERG Channels i...

XB-ART-50557

PLoS One 2015 Apr 22;104:e0123951. doi: 10.1371/journal.pone.0123951.

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

Liu QN , Trudeau MC .

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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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Species referenced: Xenopus
Genes referenced: arfgap1 arnt cfp kcnh1 kcnh2

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	Fig 2. Adenoviral i-eag domains restore slow deactivation in HEK293 cells.Representative tail current recordings from HEK293 cells expressing: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tail currents were fit with a double exponential function and the mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M; n = 6–8 cells.
	Fig 3. Adenoviral i-eag domains regulated the hERG R56Q current recorded with an AP waveform command.Representative current recordings: A (top) from WT hERG1a (black), hERG1a R56Q (red), and hERG R56Q + i-eag (blue) elicited with a voltage command mimicking a ventricular action potential A (bottom). B, Tail currents were generated by a step to −100 from 60 mV from same cells as in A). Double exponential fits (red traces) were extrapolated back to the moment of voltage change to obtain the peak tail current value (arrow). C, Box plot of peak currents elicited with AP command voltage (as in A) were normalized to peak tail current (as in B) to normalize for variations in channel expression between cells. D, Box plot of the time of the peak current elicited with the AP command voltage. For box plots, the middle line is the mean, the top and bottom lines are the 75th and 25th percentiles, respectively, and the straight lines are the 90th and 10th percentiles. n = 6–9 cells.E, Plot of currents in A) versus AP command voltage.
	Fig 4. Adenoviral i-eag domains rescued the gating-deficiencies of hERG1a(R56Q) channels expressed in hiPSC-CMs.Voltage-clamp recordings of hERG currents measured from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the protocol used. D, I-V relationships for WT hERG1a.Ad, hERG1a(R56Q).Ad and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of depolarizing step was normalized to the maximum tail current and plotted versus voltage. All values were plotted as mean ± S.E.M; n> = 15 cells.
	Fig 5. Adenoviral i-eag domains restored slow deactivation in hiPSC-CMs.Representative tail currents recorded from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tails were fit with a double exponential function, and mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M.; n> = 15 cells.
	Fig 6. hERG currents were isolated as E-4031 sensitive current.hERG currents overexpressed in hiPSC-CMs were isolated as E-4031-sensitive currents. A,B,C, The data showed families of current traces for WT hERG1a.Ad, hERG1a(R56Q).Ad and hERG1a(R56Q) + i-eag.Ad (top), after the addition of E4031(500 μM; Middle), and digitally subtracted traces to give the E-4031 sensitive currents (bottom). D, I-V relationships for each hERG channel above. The current at the end of depolarizing step was normalized to the maximum tail current and plotted versus voltage. E, Steady-state activation curves for each hERG channel above. All values were plotted as mean ± S.E.M.; n> = 15 cells.
	Fig 7. FRET between i-eag domains and hERG R56Q channels in hiPSCs.Images of cells: A, hERG1a(R56Q).Citrine.Ad; B, hERG1a(R56Q).Citrine.Ad + i-eag.CFP.Ad. C, Determination of Ratio A0. The emission spectra from hiPSCs expressing hERG1a(R56Q).Citrine was determined with excitation at 436 nm and 500 nm. Ratio A0 is the F436 spectra normalized to F500 spectra. D, Spectra method for measuring FRET and determining Ratio A. Spectra (dark blue trace) was measured from cells coexpressing i-eag.CFP.Ad + hERG1a(R56Q).Citrine.Ad by excitation at 436nm. Emission spectra of CFP (cyan) were measured in a control experiment from hiPSCs expressing i-eag.CFP. Extracted spectra (F436, red trace) is the cyan spectra subtracted from the dark blue trace and contains the emission of Citrine. Spectrum (F500, black trace) was measured from excitation of Citrine at 500 nm. Ratio A is the F436 spectra normalized to F500 spectra. E, Bar graph of Ratio A- Ratio A0, a value directly related to FRET efficiency. n = 7–9 cells. *p<0.05 for FRET between hERG1a(R56Q) and i-eag-CFP domains compared to hERG1a and i-eag-CFP control.
	Fig 8. i-eag domains regulate action potentials.Current clamp recording of action potentials from hiPSC-CMs expressing WT hERG1a.Ad, hERG1a(R56Q).Ad and hERG1a(R56Q).Ad + i-eag.Ad. A, hERG1a(R56Q) increased the action potential duration (p <0.05 compared to WT hERG1a.Ad). B, Coexpression of i-eag.Ad decreased the action potential duration in cells with hERG1a(R56Q) (p <0.05 compared to hERG1a(R56Q).Ad). C, Histogram of APD90 values in hiPSC-CMs infected by WT hERG1a.Ad, hERG1a(R56Q).Ad, and hERG1a(R56Q).Ad + i-eag.Ad. n = 12 for each. Scale bar is 50 s and 20 mV.
	Fig 1. Adenoviral i-eag domains rescue the gating-deficiencies of hERG1a(R56Q) channels expressed in HEK293 cells. A, Schematic depicting hERG channel subunit: a, WT hERG1a; b, hERG1a(R56Q); c, hERG1a(R56Q) + i-eag. B, C, D, Representative current recordings from HEK293 cells expressing: WT hERG1a.Ad, hERG1a(R56Q).Ad, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. E, IV relationships of WT hERG1a, hERG1a(R56Q), and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of the depolarizing step was normalized to the maximum tail current and plotted versus voltage. F, Representative steady-state inactivation current recording from WT hERG1a (top) using a three-pulse protocol (bottom). G, Steady-state activation and inactivation curves. Steady-state activation curves were generated by normalizing tail currents at -50 mV to the maximum tail current and plotted versus voltage. Steady-state inactivation curves were generated by normalizing the peak current and plotted versus voltage. Both steady-state activation and inactivation curves were fit with a Boltzmann function. All values were plotted as mean ± S.E.M; n = 5–13 cells.

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