Figure 4. MD simulations - binding in the fenestrations prevents fenestration gating.

(a) Side view on a TWIK-1 based TASK-1 homology model with bupivacaine located in the side fenestrations after 100 ns MD simulations. (b–c) Zoom-ins illustrating 'hit' residues of the alanine scan, which were confirmed in the MD simulations to have contacts with bupivacaine. (d) TASK-1 homology model based on TWIK-1 depicting bupivacaine within the two side fenestrations. (e) Hole analysis before (light gray) and after (gray) 100 ns MD simulations revealing a collapse of the side fenestrations when the channels move to the 'up' state. (f) Hole analysis as in (e), but in the presence of bupivacaine (purple), which prevents the movement to the 'up' state and the concomitant collapse of the fenestrations. (g) TASK-1 homology model in the absence of bupivacaine before (gray) and after 100 ns of MD simulations (black). Green arrows indicate the movement of the M2, M3 and M4 segments causing the 'down' to 'up' transition. (h) TASK-1 homology model in the presence of bupivacaine (green) before (gray) and after (purple) 100 ns, illustrating the lack of the M2, M3 and M4 movements. (i) Root-mean-square fluctuations (RMSF) calculated for the alpha carbons of the M2, M3, P2 and M4 segments in the absence and presence of bupivacaine. (j) Quantitative analysis of the bottleneck radius of the side fenestrations over the time course of 100 ns MD simulations (n = 4) in the absence (gray) and presence (purple) of bupivacaine. Data are represented as mean ± S.E.M.

Figure 4—figure supplement 1. Reduced bupivacaine inhibition by M3 mutants.

Block by 500 µM bupivacaine, analyzed for additional mutants of the M3 segment that were generated since the MD simulations predicted contacts with those residues. Inhibition was analyzed at +40 mV. Data are presented as mean ± S.E.M.. The numbers of experiments (n) are indicated within the bar graph.

Figure 4—figure supplement 2. Induced fit docking experiments predict a bupivacaine binding mode highly similar to that of classical docking experiments.

(a) For each of the four different homology models a total of 20 structures (seeds) were generated by MDs taking a frame every 0.5 ns. Induced fit dockings were performed at the 20 structures per model with 40 or 20 open fenestrations if only one fenestration is in the open state. Only in the TASK-1 model based on TWIK-1 (OO), induced fit dockings identified poses for each structure and for each fenestration (40/40; 100%). (b) Top view of the docking solution in a TWIK-1 (OO) based TASK-1 homology model with the lowest induced fit docking energy, while being compatible with the TEA versus TPA competition experiments. (c) Zoom-in of the top view from (b): induced fit dockings predict a binding model (green) similar to that proposed by classical docking experiments and MD simulations (purple) (Figure 4a–c).

Figure 4—figure supplement 3. MD simulations - binding in the fenestrations prevents fenestration gating.

(a) Hole analysis of TWIK-1 based TASK-1 homology model before (light gray) and after (gray) 100 ns MD simulation, revealing a collapse of the side fenestrations when the channels move to the 'up' state. (b) Hole analysis as in (a), but in the presence of neutral or (c) charged bupivacaine, which prevents the movement to the 'up' state and the concomitant collapse of the fenestrations.