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The hERG potassium channel intrinsic ligand regulates N- and C-terminal interactions and channel closure - PubMed

  • ️Tue Jan 01 2019

The hERG potassium channel intrinsic ligand regulates N- and C-terminal interactions and channel closure

Sara J Codding et al. J Gen Physiol. 2019.

Abstract

Human ether-à-go-go-related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an "intrinsic ligand" that is composed of residues 860-862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS-CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG "core" channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.

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Figures

Figure 1.
Figure 1.

Schemes and models of hERG channels. (A) Schematic of a single hERG channel subunit with six transmembrane (S1–S6) domains, a pore (P) domain that forms the ion conduction pathway and intracellular N- and C-terminal regions. hERG (and other KCNH channels) have an N-terminal PAS domain and a C-terminal CNBHD. The CNBHD contains an intrinsic ligand motif (orange circle). (B) Cryo-EM structure of a single hERG subunit, showing the site of the intrinsic ligand (orange) in the CNBHD (PDB accession no. 5VA2). (C) Cryo-EM structure of a hERG tetramer, showing intersubunit PAS and CNBHD interactions and showing location of intrinsic ligands (orange; PDB accession no. 5VA2). (D) X-ray crystal structure of PAS-CNBHD of KNCH1 (EAG) channels, showing intrinsic ligand (orange) at the PAS domain-CNBHD interface (PDB accession no. 4LLO). (E) Cryo-EM structure of the hERG CNBHD with the intrinsic ligand (orange; PDB accession no. 5VA2). (F) Structurally guided alignment of part of the CNBHD (β roll 5, α B helix, α C helix and the intrinsic ligand) and distal C-terminal region of KCNH family members hERG, mEAG1, and hELK1 with the CNBD of HCN2 channels. Intrinsic ligand residues are within the red box, as indicated (structural alignment: hERG, PDB accession no. 5VA2; mEAG, PDB accession no. 4LLO; and mHCN2, PDB accession no. 3ETQ; and the hELK sequence is included and primary sequence alignment is structurally guided by zElk, PDB accession no. 3UKN).

Figure 2.
Figure 2.

Mutations in the intrinsic ligand disrupted a structural interaction between the PAS domain and the CNBHD as measured with FRET spectroscopy. (A and B) Schematics of WT hERG subunit (A) and the PAS domain in trans with hERG core channels (B). (C) Line spectra plotted as intensity in arbitrary units versus wavelength (nm) from a control cell expressing WT hERG fused to citrine. Red trace (F436direct) is the emission spectra from the FRET cube, and green trace (F500) is the emission spectra from the YFP cube. Ratio A0 is the ratio of intensities of F436direct /F500 (see Materials and methods). (D) Emission spectra from positive control cell expressing PAS in trans with hERG core channels in which the dark blue trace is emission from the FRET cube, the cyan trace is a scaled control CFP spectrum, and the red trace (F436total) is calculated by subtracting the cyan from the dark blue trace. The green trace (F500) is the emission spectrum from the YFP cube. Ratio A is the ratio of intensities of F436total/F500. (E–G) Spectra from cells containing PAS domains in trans with mutant hERG core channels, PAS in trans with hERG F860A core channels (E), PAS in trans with hERG L862A core channels (F), and PAS in trans with hERG F860G,L862G core channels (G). (H) Scatter plot of relative FRET efficiency, Ratio A – Ratio A0, from data in C–G. (I) Scatter plot of Fc/Fy, which is the relative ratio of CFP emission (Fc) to citrine emission (Fy). Each point is from a different cell. The long horizontal lines are the mean and the shorter lines are the SD in H and I. Statistical significance is indicated as ***, P < 0.001 by one-way ANOVA and Tukey’s post hoc test.

Figure 3.
Figure 3.

Mutations in the intrinsic ligand disrupted PAS domain–dependent regulation of slow deactivation gating. (A and B) Scheme of hERG core channels (A) and the PAS domain in trans with hERG core channels (B). (C–E) Whole-cell voltage-clamp recordings of a family of currents from positive control hERG core channels (C), positive control PAS domain in trans with hERG core channels (D), and overlay of tail currents from C and D at −100 mV (E). (F–H) Currents from hERG F860A core channels (F), the PAS domain in trans with hERG F860A core channels (G), and overlay of tail currents from F and G (H) . (I–K) Currents from hERG L862A core channels (I), the PAS domain in trans with hERG L862A core channels (J), and overlay of tail currents from I and J (K). (L–N) Currents from hERG F860G,L862G core channels (L), the PAS domain in trans with hERG F860G,L862G core channels (M), and overlay of tail currents from L and M (N). Voltage pulses used to elicit currents from a holding potential of −80 mV in 20-mV steps to 80 mV and repolarization to −100 mV, as indicated.

Figure 4.
Figure 4.

Conductance–voltage relationships of hERG channels. Plots of conductance versus voltage for hERG core channels (black closed circles, black trace), PAS domains in trans with hERG core channels (red closed triangles, red trace), and full-length WT hERG channels for comparison (open squares, dashed trace; A); hERG F860A core channels (green closed circle, green trace) and PAS domains in trans with hERG F860A core channels (blue closed triangles, blue trace; B); hERG L862A core channels (light green closed circle, light green trace) and PAS domains in trans with hERG L862A core channels (gray closed triangles, gray trace; C); and hERG F860G,L862G core channels (magenta closed circle, magenta trace) and PAS domains in trans with hERG F860G,L862G core channels (cyan closed triangles, cyan trace; D). Traces represent fits to a Boltzmann function. Points are the mean, and error bars are the SD. n = at least three for each; see Table 1.

Figure 5.
Figure 5.

Deactivation and activation time constants. (A) Scatter plot of the fast time constant of deactivation (1) and (B) the slow time constant of deactivation (2) at −100 mV from data in Fig. 3. Deactivation was fit with a double exponential function. (C) Scatter plot of time to half-maximal activation of channels at 60 mV. ***, P < 0.001; **, P < 0.01; or *, P < 0.05 by one-way ANOVA and Tukey’s post hoc test.

Figure 6.
Figure 6.

Schematic of hERG channel PAS-CNBHD interaction and role of the intrinsic ligand. (A) WT hERG channels showing the intrinsic ligand with intrinsic ligand residues FNL in the CNBHD and the PAS-CNBHD interaction. (B) Depiction of a hERG channel with the F860G,L862G double mutant in the intrinsic ligand showing a disruption of the PAS-CNBHD interaction. Two hERG subunits (of four) are shown for clarity.

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