K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

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Science Advances  30 Oct 2020:
Vol. 6, no. 44, eabc9174
DOI: 10.1126/sciadv.abc9174
  • Fig. 1 K2P2.1 (TREK-1) selectivity filter potassium-dependent conformational changes.

    (A) Exemplar 0 and 200 mM [K+] K2P2.1 (TREK-1) SF2 2Fo-Fc electron density (1σ). Select residues and channel elements are indicated. Dashes indicate disordered regions. (B) [K+]-dependent structural changes in K2P2.1 (TREK-1) SF1 (left) and SF2 (right). Top: Superpositions of structures determined in 0 (pale yellow), 1 (yellow), 10 (light orange), 30 (yellow orange), 50 (bright orange), 100 (olive), and 200 (orange) mM [K+]. Bottom: Superposition of 0 and 200 mM [K+] structures. Dashed lines indicate regions absent from the structures. Lower panel labels mark model boundaries. (C) Exemplar 2Fo-Fc electron density (1σ) for the K2P2.1 (TREK-1):ML335 complex SF2 at 0 and 200 mM [K+]. (D) K2P2.1 (TREK-1):ML335 complex structural comparisons of SF1 (left) and SF2 (right). Top: Superposition of structures determined in 0 (blue white), 1 (pale cyan), 10 (aquamarine), 30 (light blue), 50 (marine), 100 (slate), and 200 (deep blue) mM [K+]. Bottom: Superposition of 0 and 200 mM [K+] structures. ML335 is gray and shows its molecular surface. Lower panel labels indicate the equivalent residues from (B). Potassium ions are from the 200 mM [K+] structures and are shown as magenta spheres.

  • Fig. 2 K2P2.1 (TREK-1) selectivity filter ion occupancy as a function of [K+].

    (A and B) Polder omit maps (28) for structures of (A) K2P2.1(TREK-1) and (B) K2P2.1(TREK-1):ML335 determined in 0 mM [K+] [pale yellow (5σ); blue white (4σ)], 50 mM K+ [bright orange (5σ); marine (4σ)], 100 mM [K+] [olive (4σ); slate (4σ)], and 200 mM [K+] [orange (4σ); deep blue (4σ)]. Potassium ions are magenta spheres. Sites S1 to S4 are labeled. ML335 is shown as sticks. (C and D) Potassium anomalous difference maps (29) for (C) K2P2.1(TREK-1) and (D) K2P2.1(TREK-1):ML335 determined in 1 mM [K+] [yellow (4σ); pale cyan (4σ)] and 200 mM [K+] [orange (4σ); deep blue (4σ)]. In (A) and (D), SF1 in the 200 mM [K+] conformation is shown for reference. S1 to S4 sites and select amino acids are labeled. (E) Plot of the number of observed selectivity filter ions as a function of [K+]. Colors correspond to the scheme in Fig. 1 (C and D).

  • Fig. 3 K2P2.1 (TREK-1) conductance properties and SF conformational dynamics from MD simulations.

    (A) Cumulative K+ ion permeation events over simulation time for all individual trajectories in High [K+]/+40 mV (orange) and High [K+]/+40 mV/ML335 (purple) conditions. (B) Current calculated from the trajectories in (A). Each point shows the average current from one independent trajectory; horizontal bars indicate median. (C and D) Cα RMSF values of the filter and loop regions for (C) pore domain 1 and (D) pore domain 2, for all simulated conditions. Each point represents RMSF calculated from one K2P2.1 (TREK-1) subunit of one trajectory. Conserved selectivity filter signature sequences are shaded gray. (E) PCA analysis of SF1 and SF2 dihedral angles and exemplar filter conformations. Each dot represents the instantaneous conformation of the TIGFG backbone dihedral angles from single selectivity filter. Black stars indicate the location in PC1 vs. PC2 space of the adjacent exemplar. Red dots indicate conformations immediately preceding K+ permeation events. (F) Final SF1 (left) and SF2 and loop (right) ion and backbone conformations from all simulation trajectories. Transparent gold ribbons are the final frame of each trajectory; transparent purple spheres are potassium ions. Solid blue ribbons represent the initial crystal structure conformation.

  • Fig. 4 Effects of ML335 on K2P2.1 (TREK-1).

    (A) Exemplar K2P2.1 (TREK-1) single-channel recordings at −100 mV before (left) and after (right) application of 30 μM ML335 to the same cell-attached patch. (B) Exemplar K2P2.1 (TREK-1) single-channel recordings at −100 mV in the presence of 5 μM ML335 applied in the pipette solution of a cell-attached patch. (C) Open channel probability at −100 mV from single-channel analysis calculated on recordings of ≥30-s duration. (D and E) Single-channel amplitude at (D) −100 mV and (E) +50 mV. Error bars indicate SEM (n = 5 to 7). “**” indicates P < 0.01 and “N.S.” indicates not statistically different relative to K2P2.1 (TREK-1).

  • Fig. 5 K2P SF2-M4 loop is central to C-type gate function.

    (A) K2P2.1 (TREK-1) P1-SF1-M2 (orange) and P2-SF2-M4 (slate) superposition. SF1-M2 loop (red) and SF2-M4 loop (blue) and portions having a shared conformation (dark blue) are indicated. Residue labels indicate the SF1-M2 and SF2-M4 loop ends and structural divergence point (Pro150/Ala259). (B) Sequence comparison. Arrows denote selectivity filter–outer transmembrane helix linker ends. Red indicates Pro150/Ala259 equivalents. (C) K2P2.1 (TREK-1):ML335 complex SF2-M4 loop details. SF1 (yellow), SF2 (slate), SF1-M2 (red), and SF2-M4 (marine) are indicated. Conserved Glu234 (green), Tyr270 (green), Gly260 (marine) hydrogen bond network is indicted. ML335 is shown as sticks and space filling. (D) Human K2P channel M3 glutamate network conservation (red and asterisks). Figure S7 has sequence codes. (E) Exemplar two electrode voltage clamp (TEVC) recordings at 15°C (blue), 20°C (light green), 25°C (lime green), 30°C (orange), and 35°C (red). (F) Normalized temperature responses (n ≥ 10). (G) Exemplar inside-out pressure response at 0 mmHg (black) and 50 mmHg (orange). (H) Averaged pressure responses (n ≥ 4). (I) Exemplar TEVC recordings for 30 μM ML335 (purple) activation. (J) ML335 dose-response curves (n ≥ 3). EC50 11.3 ± 3.4 and 12.7 ± 4.1 μM, maximum activation 11.9 ± 1.3, and 3.8 ± 0.4 fold for K2P2.1 (TREK1) and K2P2.1 (TREK1) Y270F, respectively. (K) Exemplar TEVC recordings for 20 μM BL-1249 (blue) activation. (L) Normalized responses to 20 μM BL-1249 (n ≥ 7). (F), (H), (J), and (L) show K2P2.1 (TREK1) (black), K2P2.1 (TREK1) Loop2sym6 (purple), K2P2.1 (TREK1) E234Q (light blue), and K2P2.1 (TREK1) Y270F (orange). “*” and “**” indicate P < 0.05 and P < 0.001, respectively.

  • Fig. 6 Structural changes associated with C-type gating.

    (A) SF1 and (B) SF2 selectivity filter changes between the active (slate) [(A) and (B) conductive] and inactive (yellow orange) [(A) pinched and (B) dilated] conformations based on the 1 mM [K+] and 0 mM [K+]:ML335 structures, respectively. Selectivity filters for 1 mM [K+] and 0 mM [K+]:ML335 show select residues. Potassium ions are magenta spheres.

Supplementary Materials

  • Supplementary Materials

    K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

    Marco Lolicato, Andrew M. Natale, Fayal Abderemane-Ali, David Crottès, Sara Capponi, Ramona Duman, Armin Wagner, John M. Rosenberg, Michael Grabe, Daniel L. Minor Jr.

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