Gustina AS and Trudeau MC (2013), The eag domain regulates hERG channel inactivat...

XB-ART-48211

J Gen Physiol 2013 Feb 01;1412:229-41. doi: 10.1085/jgp.201210870.

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The eag domain regulates hERG channel inactivation gating via a direct interaction.

Gustina AS , Trudeau MC .

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Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1-354), including the eag domain (amino acids 1-135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (Δ2-135) or both the eag domain and the adjacent proximal domain (Δ2-354) had less rectifying current-voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1-135) or the eag domain plus the proximal domain (N1-354) with N-deleted hERG Δ2-135 or hERG Δ2-354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain-containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain-containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1-354 fragments nor the presence of the proximal domain in hERG Δ2-135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG Δ2-135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

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Species referenced: Xenopus laevis
Genes referenced: cfp dtl gnao1 kcnh1 kcnh2

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	Figure 1. hERG channel schematic and confirmation of construct expression. (A) Schematic of hERG channel constructs used in this study. (B) Individual channel schematics and confocal images to confirm expression of all constructs. CFP (images on the left) was excited with a 458-nm argon ion laser, and emission was captured at 475–525 nm. Citrine (images on the right) was excited with a 488-nm argon ion laser, and emission was captured at 505–550 nm.
	Figure 2. Regulation of deactivation gating by eag domains is unaffected by the presence of the proximal domain. (A–F) Channel schematics and two-electrode voltage-clamp recordings of tail currents from hERG (A), hERG ΔN (B), hERG ΔN plus N1–135 (C), hERG Δeag (D), hERG Δeag plus N1–135 (E), and hERG ΔN plus N1–354 (F). Currents were elicited using the pulse protocol shown. Calibration bars, 2 µA and 200 ms. (G) Plot of the time constants (τ) of deactivation derived from single-exponential fits (see Materials and methods) to the tail currents in A–F. Error bars are the SEM and are within the points if not visible. n ≥ 10 for each construct.
	Figure 3. eag domain regulation of hERG rectification. (A–F) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG (A), hERG ΔN (B), hERG ΔN plus N1–135 (C), hERG Δeag (D), hERG Δeag plus N1–135 (E), and hERG ΔN plus N1–354 (F). Currents were elicited using the pulse protocol shown. Calibration bars, 1 µA and 200 ms. (G) Steady-state I-V curves derived from the currents shown in A–F by normalizing the peak outward current at the end of each depolarizing step to the extrapolated peak tail current at −100 mV and plotting versus voltage. Error bars are the SEM and are within the points if not visible. n ≥ 9 for each construct. **, P < 0.01 versus hERG.
	Figure 4. Proximal domain regulation of steady-state activation may require a covalent link to the channel. (A–F) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG (A), hERG ΔN (B), hERG ΔN plus N1–135 (C), hERG Δeag (D), hERG Δeag plus N1–135 (E), and hERG ΔN plus N1–354 (F). Currents were elicited using the pulse protocol shown. Calibration bars, 1 µA and 200 ms. (G) Steady-state activation (G-V) curves derived from the currents shown in A–F by normalizing the peak outward currents at −50 mV and plotting versus voltage. Data were fit with a Boltzmann function (see Materials and methods) to determine the V1/2 and k (slope) values. Error bars are the SEM and are within the points if not visible. n ≥ 9 for each construct.
	Figure 5. eag domain regulation of hERG recovery from inactivation. (A) Two-electrode voltage-clamp recordings of tail currents from the constructs indicated, enlarged to show the rising phase at the step to −100 mV. Currents were normalized to the peak for comparison. Calibration bar, 50 ms. (B) Plot of the time constants (τ) of recovery from inactivation derived from single-exponential fits to the rising phase of the tail currents in A from hERG (red triangle), hERG ΔN (open square), hERG ΔN plus N1–135 (closed square), hERG Δeag (open circle), hERG Δeag plus N1–135 (closed circle), and hERG ΔN plus N1–354 (closed diamond). Error bars are the SEM and are within the points if not visible. n ≥ 9 for each construct.
	Figure 6. eag domain regulation of hERG inactivation rate. (A–C) Sample two-electrode voltage-clamp recordings to isolate inactivating currents from hERG (A), hERG ΔN (B), and hERG ΔN plus N1–135 (C) using the three-pulse protocol shown. The duration of the second pulse was 20 or 5 ms based on the construct so that minimal deactivation occurred during this step. Calibration bar, 2 µA and 10 ms. (D) Plot of the time constants (τ) of inactivation derived from single-exponential fits to the current decay in A–C and from hERG Δeag (open circle), hERG Δeag plus N1–135 (closed circle), and hERG ΔN plus N1–354 (closed diamond). Error bars are the SEM and are within the points if not visible. n ≥ 9 for each construct. **, P < 0.01 versus hERG.
	Figure 7. eag domain regulation of hERG steady-state inactivation. (A–C) Sample two-electrode voltage-clamp recordings from hERG (A), hERG ΔN (B), and hERG ΔN plus N1–135 (C) using the three-pulse protocol shown. The duration of the second pulse was 20 or 5 ms based on the construct. Red arrows indicate the peak instantaneous current. Calibration bar, 2 µA and 10 ms. (D) Steady-state inactivation curves derived from the currents shown in A–C and from hERG Δeag (open circle), hERG Δeag plus N1–135 (closed circle), and hERG ΔN plus N1–354 (closed diamond) by normalizing the peak instantaneous current and plotting versus voltage (see Materials and methods). Data were fit with a Boltzmann function to determine the V1/2 and k (slope) values. Error bars are the SEM and are within the points if not visible. n ≥ 8 for each construct.
	Figure 8. The eag domain regulates the timing of the peak hERG resurgent current. (A) Representative current recordings of hERG (red), hERG ΔN (black), hERG ΔN plus N1–135 (purple), hERG Δeag (blue), hERG Δeag plus N1–135 (light blue), and hERG ΔN plus N1–354 (green) in response to a dynamic ramp clamp. Currents were normalized to the extrapolated peak tail at −100 mV (see Materials and methods). Bar, 50 ms. (B) Graph of the normalized current from A versus the voltage during the current ramp. (C) Box plot of the voltage at the current peak in A and B for the constructs indicated. (D) Box plot of the time to the current peak in A and B for the constructs indicated. (E) Box plot of the peak inward current at −100 mV derived from A and B for the constructs indicated. The middle line of the box is the mean, the top and bottom are the 75th and 25th percentiles, and the vertical lines indicate the 90th and 10th percentiles. n ≥ 10 for each construct. **, P < 0.01 versus hERG and versus hERG and hERG ΔN.
	Figure 9. Model of eag domain interaction with the channel. (A) Schematic of WT hERG showing eag domain interaction with the CNBHD and a proximal domain, which is continuous with the transmembrane regions. (B) Schematic of hERG Δeag plus N1–135 showing eag domain interaction with the CNBHD and a proximal domain, which is continuous with the transmembrane regions but not attached to the eag domain. (C) Schematic of hERG ΔN plus N1–354 showing eag domain interaction with the CNBHD and a proximal domain, which is continuous with the eag domain but not connected to the transmembrane regions.

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