Gating and regulation of KCNH (ERG, EAG, and ELK) channels by intracellular domains

Figures

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  Figure 1.. Cladogram of channels in the KCNH family. Comparison of ERG (KCNH2), EAG (KCNH1), ELK (KCNH3), CNG, HCN and Kv (KCNMA) channels.

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  Figure 2.. Functional characteristics of KCNH channel currents. Schematic drawings of characteristic currents from A) ERG, B) EAG and C) ELK channels. Schematic of voltage-activation relationships for D) ERG, E) EAG and F) ELK channels.

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  Figure 3.. KCNH subunits. (a) Linear scheme of KCNH channel subunit, (b) Topology of KCNH channel subunit (c) Ribbon diagram of single hERG subunit (PDB 5VA2) and (d) ribbon diagram of single EAG channel subunit (PDB 5K7L). PAS-CAP = light green, PAS = green, N-linker = pink, Voltage sensing domain (VSD) = gray, Pore domain = black, C-linker = light blue, CNBHD = blue, Intrinsic ligand = orange, distal C-terminal domain = brown.

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  Figure 4.. Non-domain-swapped VSD and Pore Domains in KCNH tetramers. Schematic of top-down view of (a) a representative KCNH tetramer and (b) a representative Kv channel tetramer. Top-down view of (c) hERG tetramer CryoEM structure and (d) Kv1.2 tetramer X-ray crystallography structure (PDB 3LUT). Each individual subunit in A-D is a different color to highlight non-domain swapping versus domain-swapping. Ribbon diagram of one channel subunit in C and D overlays the space-filling depiction of one subunit of the tetramer. The S4-S5 linker in C,D is depicted as cyan.

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  Figure 5.. KCNH activation gates and Ca-CaM association with KCNH1. (a) Side view of overlay of closed EAG activation gate (light blue) and open hERG activation gate (black). Dashed lines depict distances between the Q476 residues in the lower S6 domain in EAG, which result in a pore radius of less than 1 angstrom and the Q664 residues in the lower S6 of hERG, which result in a pore radius of approximately 5 angstroms. (b) Schematic of top-down view of KCNH1 channels associated with four Ca-CaM proteins (purple) indicating intersubunit interaction with the channel. (c) Bottom-up view of KCNH1 and Ca-CaM (purple) indicating that the N lobe binds the N-linker domain (pink) distal to the PAS domain (green) and the C lobe binds the C-terminal region (brown) distal to the CNBHD (blue) from an adjacent subunit. The transmembrane domains and most of the C-linker were removed for clarity. (d) Side view of KCNH1 with three (of four) Ca-CaMs. Same colors as in C plus the addition of the C- linker (light blue), VSD (gray) and Pore domain (black).

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  Figure 6.. Direct PAS and CNBHD interaction in KCNH channels. (a) Top-down schematic of KCNH tetramer showing intersubunit PAS-CNBHD interaction. Single subunits are depicted as the same color. (b) Side view of hERG tetramer with one subunit shown as a ribbon to indicate that the PAS is located below the VSD and Pore of the same subunit and the CNBHD not near the PAS of the same subunit. (c) Top-down view of hERG tetramer indicating that a PAS domain from one subunit (i.e. encircled for the green subunit) interacts with the CNBHD of an adjacent subunit (i.e. encircled for the blue subunit). The transmembrane domains were removed for clarity. (d) X-ray crystallography structure of the PAS (green) and CNBHD (blue) of EAG channels in isolation from the transmembrane domains (PDB 4LLO). Intrinsic ligand (orange) in the CNBHD. Side chains R57 (green sticks) in the PAS domain and D642 (blue sticks) in the CNBHD form a salt bridge.

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  Figure 7.. The intrinsic ligand of hERG channels (a) High-resolution X-ray crystal structure of the hERG CNBHD (PDB 6SYG) colored by secondary structure (red = β sheet, cyan = α-helix, magenta = loop). Three amino acids (F860, N861 and L862) form the core of the intrinsic ligand (orange). (b) The intrinsic ligand (orange) sits within a meshwork of interacting residues, labeled by number, and colored by atom, C = gray, N = blue, O = red, black dashed lines = H-bond. A salt-bridge (green-dashed line) between E807 and R863. (c) Alignment of C-terminal region of KCNH and HCN channels, underline = h bond network, red text = salt bridge, yellow highlight = conserved with respect to KCNH2, gold box = intrinsic ligand, secondary structure indicated by red arrow (β-sheets 4 and 5) and blue cylinder (α-helixes B and C).

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  Figure 8.. The intrinsic ligand is necessary for the PAS-CNBHD interaction. Scheme of (a) hERG FRET sensor with in line donor (Cerulean) and acceptor (Venus) fluorophores and robust FRET (red arrow), (b) mutation at F860 in the intrinsic ligand reduces FRET (smaller red arrow), (c) hERG FRET sensor with in trans donor (CFP) and acceptor (Citrine) fluorophores in which the PAS-CFP and hERG delta PAS-Citrine channel are co-expressed in trans. Robust FRET was detected (red arrow) (d) mutation of F860 to G and L862 to G abolishes FRET.

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  Figure 9.. Summary of PAS-CNBHD interactions and regulators in KCNH channels. Scheme of 2 (of 4) subunits of a KCNH channel. Major domains are the VSDs (gray), Pore domains (black), PAS-CAP (light green), N-linker (pink), C-linker (light blue) and distal C-terminal region (brown). Intersubunit PAS (green) and CNBHD (dark blue) interaction is depicted with the intrinsic ligand (orange) at the surface of the CNBHD. ScFV fragments target the hERG PAS domain and impair gating and chlorpromazine binds to the EAG channel PAS and regulates gating. Flavonoids and voltage regulate the intrinsic ligand. Calcium-Calmodulin targets sites close to the PAS and CNBHD in EAG channels and inhibits channel function, and voltage and PIP2 regulate ELK channel PAS-CNBHD interactions.