A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore

Abstract

Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.

FIGURE 1.

A1899 selectively blocks human TASK-1 channels expressed in Xenopus oocytes. A, human TASK-1 channels recorded with a voltage step protocol (200-ms steps from −70 mV to +70 mV with an increment of 10 mV; holding potential, −80 mV) before (left) and after (right) application of 40 nm A1899 (see inset for chemical structure of A1899). B, dose-response curve of A1899 on human TASK-1. Block was analyzed at the end of the test pulse to +40 mV. C, test for sensitivity of different potassium channels to 100 nm A1899 after heterologous expression in Xenopus oocytes. D, I-V relationships for TASK-1 in bath solution with high potassium concentration before (□) and after (■) application of 400 nm A1899. E, the percentage of inhibition by 400 nm A1899 from D plotted against the applied voltage. F, application of different concentrations of A1899 to TASK-3 channels recorded in intact whole oocytes (□) and in inside-out macropatches (○). The inset shows the recording of inside-out macropatches with a higher temporal resolution. TEVC, two-electrode voltage clamp.

FIGURE 2.

M2 and M4 segments of TASK-1 are part of the binding site for A1899. Four different chimeras, with TASK-4 transmembrane segments introduced into a TASK-1 background replacing M1, M2, M3, or M4, were analyzed for their IC₅₀ of block by A1899. hTASK, human TASK.

FIGURE 3.

The HRE influences A1899 binding. A, the sequence of the HREs of TASK-1 and TASK-3 differ in one amino acid (VLRF(M/L)T) (indicated by a box). In both channels, in-frame deletion of the HRE (bottom) leads to a massive increase in IC₅₀ values. rTASK, rat TASK. B, dose-response curves of A1899 on human TASK-1 WT (■) and an M247L (▴) mutant.

FIGURE 4.

The A1899 binding site includes residues of the M2 and M4 segment and the selectivity filter. A–F, mutant TASK-1 channels were expressed in Xenopus oocytes, and block by 400 nm A1899 was analyzed. A and D, sample current traces for wild-type TASK-1 and mutated channels before (black) and after (gray) application of 400 nm A1899. B, C, E, and F, mutant TASK-1 channels with a reduced sensitivity to A1899 indicate residues as part of the binding site for A1899 (illustrated as striated bars). Threonine residues of the P1 and P2 pore loops are part of the A1899 binding site. In the M2 and M4 segment, residues of the binding site are located at a distance of 3–4 amino acids.

FIGURE 5.

TASK-1 channels with multiple mutations at the drug binding site show a strong reduction of A1899 sensitivity. A, sample traces of TASK-1 wild-type, double, and triple mutant channels before (black) and after (gray) application of 400 nm A1899. B, inhibition of TASK-1 channels with multiple mutations by 400 nm A1899.

FIGURE 6.

Open-state pore homology model of TASK-1 based on the KvAP structure. The figure illustrates residues identified as pore-facing using A1899. For better display, the backbone of half a TASK-1 subunit (M1, P1 and M2) is hidden, whereas the identified residues are still depicted. The backbone is shown transparent for a better view on the residues of the drug binding site.

FIGURE 7.

Docking of A1899 to an open-state pore homology model of TASK-1. The binding mode of A1899 in the pore of TASK-1 as predicted by docking using the software GOLD is shown. All amino acids predicted to interact with the drug molecule are illustrated. For a better display of A1899 binding, the TASK-1 channel is shown in two orientations (rotated by 90°). The left panel shows the two M1, P1, and M2 segments, and the right panel shows the two M3, P2, and M4 segments. H-bonds are depicted as dotted lines between A1899 and the threonine residues of the selectivity filter.

FIGURE 8.

Molecular dynamics simulations validate the A1899 binding site. For a better display of A1899 binding, the TASK-1 channel is shown in two orientations (rotated by 90°). The left panel shows the two M1, P1, and M2 segments, and the right panel shows the two M3, P2, and M4 segments. All amino acids predicted to have multiple contacts with the drug molecule are illustrated. In addition, A1899 interacts with the potassium ion at S₄ of the selectivity filter.