Structures of TASK-1 and TASK-3 K2P channels provide insight into their gating and dysfunction in disease

Figures

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  Figure 1. CryoEM structure of TASK-1. a. Cartoon showing the topology of a single TASK-1 subunit indicating the relative position of the pH-sensor, H98 (red dot), the M1-M4 transmembrane helices, and the X-gate. b. Cryo-EM structure of TASK-1, with chain A in green and chain B in pink. c. Superimposition of this TASK-1 cryoEM structure and the TASK-1 crystal structure 6RV2 (dark grey) d. RMSD plot of the TASK-1 cryoEM structure compared to the crystal structure (6RV2). e. H98 in TASK-1 at pH 8.5 from PDB:6RV2. f. Conformation of H98 at pH 7.5 as determined in this cryoEM structure. In this structure the H-bonding between Q77 and Q209 is disrupted. g. Dual rotamer density of H98 with the upwards conformation shown and density for the down conformation clearly visible.

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  Figure 2. CryoEM structure of TASK-3. a. Cartoon showing the structure of TASK-3, with chain A in gold and chain B in teal. b. The X-gate environment of TASK-3. c. Pore radius profile of WT TASK-3 and the other structures determined in this study. d. H98 downward conformation. e. RMSD plot of TASK-3 versus TASK-1. f. Whole-cell recordings of WT TASK-3 (n=95) and TASK-3 with ‘DDSA’ mutations: G129D (n=42), N133S (n=60), and L241F (n=51). ***p <0.0001. g. Current-voltage responses measured in inside-out patches from Xenopus oocytes expressing WT or L244A mutant TASK-3 channels in symmetrical high K+ at pH 7.4 in the absence (black) and in the presence of 1 µM TPenA (teal). Currents were elicited by the voltage steps indicated above the traces. The dotted red line indicates the extent of block by TPenA seen in WT TASK-1 when the X-gate opens upon voltage-activation. This is not seen in the more active L244A mutant channel. The example measurements shown here for WT and mutant channels were repeated with similar results (n=6).

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  Figure 3. Cryo-EM structure of TASK-3 G236R. a. Cartoon showing the structure of TASK-3 G236R, with the two chains of the homodimer shown in blue. b. The position of R236 within the inner cavity is shown as orange VdW spheres. c, d. Overlay of TASK-3 (gray) and TASK-3 G236R (blue) highlighting the additional movement in the X-gate and increased pore dilation in that region (expanded in panel d).

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  Figure 4. Comparison of pH-dependent changes in the filters of TASK and TWIK channels. a. Top down view of TASK-1 at pH 7.5, H98 pointing upwards, Y96 of the GYG motif is in its canonical orientation, b. TASK-3 at pH 7.5, H98 pointing down, Y96 in canonical position. c. TASK-3 pH 6.0 low K+, H98 reoriented downward, with Y96 and F202 pointing upwards blocking the pore, d. For Comparison, TWIK-1 at pH 5.5 is also shown with major rearrangement and distortion of the filter loop that restricts the pore, pH-sensor H122 is also pointing up. e. Schematic side view of the filter region summarising the sequence of possible events involved pH-dependent gating of TASK channels. Initially, at high pH the filter is in a canonical, fully conductive conformation represented by full ion occupancy of the filter. In response to increasing [H+], the first step involves protonation of H98 and upwards flipping of this side chain. Consequent changes in H-bonding behind the filter and the structural dynamics of this region may therefore begin to reduce K+ permeation and represent the first step in pH-gating. However, at even lower pH and low [K+], more dramatic structural changes have also been observed that…