Gustina AS and Trudeau MC (2011), hERG potassium channel gating is mediated by N-...

XB-ART-43235

J Gen Physiol 2011 Mar 01;1373:315-25. doi: 10.1085/jgp.201010582.

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hERG potassium channel gating is mediated by N- and C-terminal region interactions.

Gustina AS , Trudeau MC .

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Human ether-á-go-go-related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide-binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain-deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)-PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain-deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD.

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Species referenced: Xenopus laevis
Genes referenced: arnt gnao1 kcnh2 mia3

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	Figure 1. hERG activation gating is altered by C-terminal CNBD deletion. (A) Schematic of the hERG K+ channel with relevant N- and C-terminal domains indicated. (B–F) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG (B), hERG ΔN (C), hERG Δdistal C (D), hERG ΔCNBD/distal C (E), and hERG ΔCNBD (F). Currents were elicited using the pulse protocols indicated. Insets showing tail currents at −50 mV are 200 ms in duration; inset scale bars are 0.5 μA. (G) I-V relationship for B–F. The currents at the end of each depolarizing pulse were normalized to the peak current for that cell and plotted versus voltage. n ≥ 4 for each. (H) The steady-state voltage dependence of activation (G-V) for B–F. The instantaneous tail currents at −50 mV were normalized to the peak instantaneous tail current for that cell and plotted versus voltage. n ≥ 5 for each. Data were fit with a Boltzmann function to determine the V1/2 and k (slope) values. (I) Plot of the time to half-maximal activation versus voltage for B–F. n ≥ 4 for each. For G–I, the values are plotted as the means ± SEM. The error bars are within the points if not visible.
	Figure 2. C-terminal CNBD deletion affects hERG deactivation. (A–E) Channel schematics and two-electrode voltage-clamp recordings of a family of tail currents from hERG (A), hERG ΔN (B), hERG Δdistal C (C), hERG ΔCNBD/distal C (D), and hERG ΔCNBD (E). Currents were elicited using the pulse protocol indicated. (F) Plot of the time constants (τ) of deactivation derived from a single exponential fit (see Materials and methods) to the tail currents in A–E. The values are plotted as the means ± SEM. The error bars are within the points if not visible. n ≥ 5 for each.
	Figure 3. N-terminal region deletion does not further alter gating in C-terminal–deleted channels that include a CNBD deletion. (A, C, and E) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG ΔN Δdistal C (A), hERG ΔN ΔCNBD/distal C (C), and hERG ΔN ΔCNBD (E). Currents were elicited using the pulse protocols indicated. Insets showing tail currents at −50 mV are 200 ms in duration; inset scale bars are 0.5 μA. (B, D, and F) Two-electrode voltage-clamp recordings of a family of tail currents from the constructs in A, C, and E. Currents were elicited using the pulse protocol indicated. (G) Normalized I-V relationship for A, C, and E. n ≥ 6 for each. (H) Normalized G-V relationship for A, C, and E. Data were fit with a Boltzmann function to determine the V1/2 and k (slope) values. n ≥ 6 for each. (I) Plot of the time to half-maximal activation for A, C, and E. n ≥ 6 for each. (J) Plot of the time constants (τ) of deactivation derived from a single exponential fit to the tail currents in B, D, and F. n ≥ 5 for each. For G–J, the values are plotted as the means ± SEM. The error bars are within the points if not visible. hERG (closed squares) and hERG ΔN (open squares) are included on all plots for reference.
	Figure 4. N1–135 coexpression has no effect on channels that have a CNBD deletion but does modify gating in distal C-terminal region–deleted channels. (A, C, and E) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG ΔN Δdistal C + N1–135 (A), hERG ΔN ΔCNBD/distal C + N1–135 (C), and hERG ΔN ΔCNBD + N1–135 (E). Currents were elicited using the pulse protocols indicated. Insets showing tail currents at −50 mV are 200 ms in duration; inset scale bars are 0.5 μA. (B, D, and F) Two-electrode voltage-clamp recordings of a family of tail currents from the constructs in A, C, and E. Currents were elicited using the pulse protocol indicated. (G) Normalized I-V relationship for A, C, and E. n ≥ 5 for each. (H) Normalized G-V relationship for A, C, and E. Data were fit with a Boltzmann function to determine the V1/2 and k (slope) values. n ≥ 6 for each. (I) Plot of the time to half-maximal activation for A, C, and E. n ≥ 5 for each. (J) Plot of the time constants (τ) of deactivation derived from a single exponential fit to the tail currents in B, D, and F. n ≥ 5 for each. For G–J, the values are plotted as the means ± SEM. The error bars are within the points if not visible. hERG (closed squares) and hERG ΔN (open squares) are included on all plots for reference.
	Figure 5. Biochemical interaction of hERG 1–135 with the CNBD. (A) Schematic of the hERG channel and the constructs used for the biochemical interaction assay. (B) Coomassie stain (CB, Coomassie blue) and Western blot of GST N1–135 and GST-only control probed with α-Flag to detect bound hERG H6 C-linker/CNBD-Flag.
	Figure 6. Coexpression of N-terminal region–deleted hERG with C-terminal region–deleted hERG partially compensates for gating alterations caused by either N- or C-terminal region deletion. (A and C) Channel schematics and two-electrode voltage-clamp recordings of a family of currents from hERG ΔpCNBD/distal C (A) and hERG ΔN + hERG ΔpCNBD/distal C (C). Currents were elicited using the pulse protocols indicated. Insets showing tail currents at −50 mV are 200 ms in duration; inset scale bars are 0.5 μA. (B and D) Two-electrode voltage-clamp recordings of a family of tail currents from the constructs in A and C. Currents were elicited using the pulse protocol indicated. (E) Normalized I-V relationship for A and C. n ≥ 5 for each. (F) Normalized G-V relationship for A and C. Data were fit with a Boltzmann function to determine the V1/2 and k (slope) values. n ≥ 6 for each. (G) Plot of the time to half-maximal activation for A and C. n ≥ 5 for each. (H) Plot of the time constants (τ) of deactivation derived from a single exponential fit to the tail currents in B and D. n ≥ 5 for each. For E–H, the values are the means ± SEM. The error bars are within the points if not visible. hERG (closed squares) and hERG ΔN (open squares) are included on all plots for reference.
	Figure 7. Model of N- and C-terminal region interactions in the hERG K+ channel. (A) Interaction of the N-terminal PAS domain with the C-terminal CNBD within the same subunit of the hERG channel. (B) Interaction of the PAS domain with the CNBD on adjacent subunits of the hERG channel.

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