Gustina AS and Trudeau MC (2009), A recombinant N-terminal domain fully restores ...

XB-ART-40437

Proc Natl Acad Sci U S A 2009 Aug 04;10631:13082-7. doi: 10.1073/pnas.0900180106.

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A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels.

Gustina AS , Trudeau MC .

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Human ether á go-go related gene (hERG) potassium channels play a central role in cardiac repolarization where channel closing (deactivation) regulates current density during action potentials. Consequently, mutations in hERG that perturb deactivation are linked to long QT syndrome (LQTS), a catastrophic cardiac arrhythmia. Interactions between an N-terminal domain and the pore-forming "core" of the channel were proposed to regulate deactivation, however, despite its central importance the mechanistic basis for deactivation is unclear. Here, to more directly examine the mechanism for regulation of deactivation, we genetically fused N-terminal domains to fluorescent proteins and tested channel function with electrophysiology and protein interactions with Förster resonance energy transfer (FRET) spectroscopy. Truncation of hERG N-terminal regions markedly sped deactivation, and here we report that reapplication of gene fragments encoding N-terminal residues 1-135 (the "eag domain") was sufficient to restore regulation of deactivation. We show that fluorophore-tagged eag domains and N-truncated channels were in close proximity at the plasma membrane as determined with FRET. The eag domains with Y43A or R56Q (a LQTS locus) mutations showed less regulation of deactivation and less FRET, whereas eag domains restored regulation of deactivation gating to full-length Y43A or R56Q channels and showed FRET. This study demonstrates that direct, noncovalent interactions between the eag domain and the channel core were sufficient to regulate deactivation gating, that an LQTS mutation perturbed physical interactions between the eag domain and the channel, and that small molecules such as the eag domain represent a novel method for restoring function to channels with disease-causing mutations.

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Species referenced: Xenopus
Genes referenced: kcnh1 kcnh2
GO keywords: potassium channel activity [+]

???displayArticle.disOnts??? short QT syndrome [+]
???displayArticle.omims??? LONG QT SYNDROME 1; LQT1 [+]

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	Fig. 1. Regulation of deactivation gating by a recombinant N-terminal eag domain. (A) Schematic of hERG channel constructs. Two-electrode voltage-clamp recordings of a family of tail currents from (B) hERG YFP (closed squares), (C) hERG ΔN YFP (open squares) and (D) N1–135 CFP + hERG ΔN YFP (closed circles). Currents were elicited by the voltage pulse protocol indicated. Calibration bars, 1 μA and 250 ms. (E) Plots of the time constants (τ) derived from a single exponential fit (see Materials and Methods) to the tail currents in (B–D). Plots also include CFP + hERG ΔN YFP (open circles). Error bars are the SEM and are inside the points when not visible. n ≥ 6 for each. (F) Current traces from N-truncated hERG channels, eag domains: N-truncated hERG channels at the indicated ratios and wild-type hERG scaled to the peak at −100 mV for comparison. Zero current given by dotted line. Scale bar, 100 ms. (G) Box plot of time constants of deactivation, in which the middle line of the box is the mean, the top and bottom are the 75th and 25th percentiles, and the straight lines are the 90th and 10th percentiles. n ≥ 5 for each.
	Fig. 2. Proximity of the soluble hERG N-terminal eag domain and the core of the hERG channel measured using FRET. (A) Spectral method for measuring FRET and determining Ratio A. Spectra (red trace) were measured from cells coexpressing N1–135 CFP + hERG ΔN S620T YFP by excitation at 458 nm. Emission spectra of CFP (cyan) measured in a control experiment from oocytes expressing N1–135 CFP. Extracted spectra (F458, green trace) is the cyan spectra subtracted from the red spectra and contains the emission of YFP. Spectra (F488, black trace) from excitation of YFP by a 488-nm laser line. Ratio A is the F458 spectra normalized to F488 spectra. (B) Plot of Ratio A (red line) versus wavelength. (C) Determination of Ratio A0. As a control, the emission spectra from oocytes expressing hERG ΔN S620T YFP channels was determined with excitation with a 458 laser line (F458, red trace) and with excitation of a 488 laser line (F488, black trace). Ratio A0 is the F458 spectra normalized to F488 spectra. (D) Plot of Ratio A0 (red line) versus emission wavelength. (E) Bar graph of Ratio A − Ratio A0, a value directly related to FRET efficiency, for the indicated channels and bar graph of ratio of CFP intensity to YFP intensity (Fc/Fy). SEM is denoted by error bars; the number of experiments (n) is indicated in parentheses.
	Fig. 3. Reduced regulation of deactivation gating by soluble eag domains containing point mutations. Two-electrode voltage-clamp recordings of ionic current from (A) N1–135 (Y43A) CFP fragment + hERG ΔN YFP channels (inverted, closed triangle) and (B) N1–135 (R56Q) CFP fragment + hERG ΔN YFP channels (closed triangle). Calibration bar is 2 μA and 250 ms. (C) Plot of time constant (τ) of deactivation versus voltage. Time constants for N1–135 CFP + hERG ΔN YFP (closed circles) and hERG ΔN YFP (open squares) included for comparison. Error bars are the SEM and the n ≥ 3 for each point. (D) Bar graph of relative FRET efficiency (Ratio A − Ratio A0) and ratio of CFP to YFP (Fc/Fy) for the indicated channels. SEM is denoted by error bars; the number of experiments (n) is indicated in parentheses.
	Fig. 4. Regulation of deactivation gating in a hERG channel with an LQTS mutation by soluble eag domains. Two-electrode voltage-clamp recordings of ionic currents from (A) hERG Y43A (closed inverted triangle), (B) N1–135 CFP + hERG Y43A (open inverted triangle), (C) hERG R56Q (closed triangle), (D) N1–135 CFP + hERG R56Q (open triangle), and (E) N1–135 CFP + hERG YFP (diamond) elicited with the voltage pulse as indicated. Calibration bars are 2 μA and 250 ms. (F) Plot of deactivation time constant (τ). hERG YFP (closed squares) and hERG ΔN YFP (open squares) are included as a reference. Error bars are the SEM and the n ≥ 3 for each point. (G) Bar graph of relative FRET efficiency (Ratio A − Ratio A0) and ratio of CFP to YFP (Fc/Fy) for the indicated channels. SEM is denoted by error bars; the number of experiments (n) is indicated in parentheses.
	Fig. 5. Schematic for function of eag domain. (A) Interaction of the N-terminal eag domain with other regions of the hERG channel. (B) Interaction of the eag domain with the channel is noncovalent and does not require the proximal N-terminal region. (C) Soluble eag domains do not supplant eag domain in wild-type channels. (D) Soluble eag domains supplant eag domains with point mutations, indicating that the mutation weakened an eag domain–channel interaction.

References [+] :

Al-Owais, Role of intracellular domains in the function of the herg potassium channel. 2009, Pubmed