An allosteric ligand-binding site in the extracellular cap of K2P channels

Abstract

Two-pore domain potassium (K2P) channels generate leak currents that are responsible for the maintenance of the resting membrane potential, and they are thus potential drug targets for treating diseases. Here, we identify N-(4-cholorphenyl)-N-(2-(3,4-dihydrosioquinolin-2(1H)-yl)-2-oxoethyl)methanesulfonamide (TKDC) as an inhibitor of the TREK subfamily, including TREK-1, TREK-2 and TRAAK channels. Using TKDC as a chemical probe, a study combining computations, mutagenesis and electrophysiology reveals a K2P allosteric ligand-binding site located in the extracellular cap of the channels. Molecular dynamics simulations suggest that ligand-induced allosteric conformational transitions lead to blockage of the ion conductive pathway. Using virtual screening approach, we identify other inhibitors targeting the extracellular allosteric ligand-binding site of these channels. Overall, our results suggest that the allosteric site at the extracellular cap of the K2P channels might be a promising drug target for these membrane proteins.TREKs are members of the two-pore domain potassium (K2P) channels, being important clinical targets. Here the authors identify inhibitors of K2P that bind to an allosteric site located in their extracellular cap, suggesting that it might be a promising drug target for these channels.

Fig. 1

Inhibition of TREK subfamily channels by TKDC in CHO cells. a Chemical structure of TKDC. b, c Typical whole-cell current traces recorded from CHO cells overexpressing the TREK-1 channel with 10 μM TKDC b or DMSO application c. Currents were elicited by depolarizing voltage steps from a holding potential of −80 mV to + 80 mV in 20 mV increments, followed by stepping down to −60 mV. d Dose-dependent inhibition of TKDC on TREK-1, TREK-2 and TRAAK channels. e The statistics of the half-inhibitory concentrations of TKDC for TREK-1 (n = 7), TREK-2 (n = 7), and TRAAK (n = 8) channels. IC₅₀ values were obtained via dose–response fitting. The Kruskal–Wallis test was used for statistical analysis; ** indicates P < 0.01. The data are shown as the mean ± s.e.m

Fig. 2

Binding mode of TKDC to TREK-1. a Binding site of TKDC in the extracellular cap of the TREK-1 channel. b Detailed view of the interactions between TKDC and the TREK-1 channel. TKDC and the residues involved in binding are shown as sticks. The protein is shown in a cartoon and surface depiction. c Dose-dependent inhibition of TKDC on the WT and mutant TREK-1 channels. d Histograms summarizing the half-inhibitory concentrations for WT (n = 7), L102A (n = 10), Q76A (n = 6) and I80A (n = 3) mutant TREK-1 channels. IC₅₀ values were obtained via dose–response fitting. The Kruskal–Wallis test was used for statistical analysis; * indicates P < 0.05, ** indicates P < 0.01. The data are shown as the mean ± s.e.m

Fig. 3

Substitutions of key residues in the extracellular cap of TRAAK channel. a Multiple sequence alignment for the extracellular caps of TREK-1, TREK-2, and TRAAK channels. b Dose-dependent inhibition of TKDC on the WT and mutant TRAAK channels. c Histograms summarizing the half-inhibitory concentrations of TKDC for the WT (n = 8), A35Q (n = 8), E38T (n = 6) and V42Q (n = 7) mutant TRAAK channels. Mutations of these residues in the extracellular cap showed enhanced inhibitory effects of TKDC. IC₅₀ values were obtained via dose–response fitting. One-way ANOVA with post hoc LSD test was used for statistical analysis [F (3, 20) = 18.551]; ** indicates P < 0.01. The data are shown as the mean ± s.e.m. d, e Docking modes of TKDC in A35Q TRAAK d, in V42Q TRAAK e, and in E38T TRAAK f. The protein is shown as a cartoon. TKDC and key residues are shown as sticks

Fig. 4

Inhibition of TREK subfamily channels by 28NH in CHO cells. a Chemical structure of 28NH. b Dose-dependent inhibition of TRAAK by TKDC and 28NH. c The statistics of IC₅₀ values for the inhibition of TREK-1, TREK-2 and TRAAK channels by TKDC and 28NH. IC₅₀ values were obtained via dose–response fitting. The unpaired t-test was used for statistical analysis [t (7) = 1.027 and n = 9 for TREK-1, t (4) = 0.910 and n = 6 for TREK-2, and t (6) = 5.724 and n = 8 for TRAAK]; ** indicates P < 0.01. The numbers in the bars indicate the number of cells studied per condition. The data are shown as the mean ± s.e.m. d Binding model of 28NH to TRAAK. The protein is shown as a cartoon. 28NH and key residues are shown as sticks

Fig. 5

Different conformations of TREK-1 obtained from MD simulations. a Conformation of TREK-1 in the apo system S_apo. b Conformation of TREK-1 in the complex system S_complex with TKDC. The protein is shown as a cartoon. The extracellular E2 helix (cyan) and the selectivity filter below (green and yellow) are shown as surfaces. The filter in green and the E2 helix in cyan are in the same subunit. The yellow filter is in the other subunit. TKDC is shown as sticks. The red dashed arrow represents the conductive pathway. The red solid arrows indicate the movement of the E2 helix under the influence of TKDC. All illustrations are from the end of simulation runs

Fig. 6

Inhibitors of TREK-1 identified in structure-based virtual screening. a Cartoon showing superimposed binding models of 25 hits. b Chemical structures of the discovered inhibitors of TREK-1. c Dose-dependent inhibition of TREK-1 by TKN1 and TKN2. IC₅₀ values were obtained via dose–response fitting

Fig. 7

Chronic administration of TKDC in mice. a Time spent immobile in the forced swimming test after administration of TKDC and fluoxetine (FLX) [one-way ANOVA with post hoc LSD test, F (4,55) = 4.20]. Veh indicates the vehicle-treatment group. TKDC was administered at doses of 0.5, 1 and 5 mg kg⁻¹. Fluoxetine was administered at a dose of 10 mg kg⁻¹. b Time spent immobile in the tail suspension test after administration of TKDC and fluoxetine [one-way ANOVA with post hoc LSD test, F (4,53) = 2.55]. c Percentage of distance traveled in the center of the field over the total distance traveled after administration of TKDC and fluoxetine in the open field test (one-way ANOVA with post hoc LSD test, F (4,51) = 3.81). d Total distance traveled after administration of TKDC and fluoxetine in the open field test (one-way ANOVA with post hoc LSD test, F (4,51) = 2.02). The numbers in the bars indicate the number of cells studied per condition. The results are shown as the mean ± s.e.m; * indicates P < 0.05, ** indicates P < 0.01