Structure of the human K2P13.1(THIK-1) channel reveals a novel hydrophilic pore restriction and lipid cofactor site

Abstract

The halothane-inhibited K_2P leak potassium channel K_2P13.1 (THIK-1)^1-3 is found in diverse cells^1,4 including neurons^1,5 and microglia^6-8 where it affects surveillance6, synaptic pruning7, phagocytosis7, and inflammasome-mediated interleukin-1β release^6,8,9. As with many K_2Ps^1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members^3,15,16, polyunsaturated fatty acid (PUFA) lipids modulate K_2P13.1 (THIK-1)^1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K_2P13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K_2Ps^13,18-24, K_2P13.1 (THIK-1) has a two-chamber aqueous inner cavity obstructed by a M4 transmembrane helix tyrosine (Tyr273, the flow restrictor). This hydrophilic barrier can be opened by an activatory mutation, S136P²⁵, at natural break in the M2 transmembrane helix and by intrinsic channel dynamics. The structures also reveal a buried lipid in the P1/M4 intersubunit interface at a location, the PUFA site, that coincides with the TREK subfamily K_2P modulator pocket for small molecule agonists^18,26,27. This overlap, together with the effects of mutation on K_2P13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K_2P13.1 (THIK-1) cofactors. Comparison with the PUFA-responsive VGIC Kv7.1 (KCNQ1)^28-31 reveals a shared role for the equivalent pore domain intersubunit interface in lipid modulation, providing a framework for dissecting the effects of PUFAs on the VGIC superfamily. Our findings reveal the unique architecture underlying K_2P13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K_2Ps by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation^6,32 and autism⁶.

Keywords: K2P channel; cryo-electronmicroscopy; electrophysiology; lipid nanodiscs; polyunsaturated fatty acid (PUFA).

Figure 1. K_2P13.1(THIK-1) structural features

a, K_2P13.1(THIK-1) cartoon diagram (salmon and deep teal) showing side (left and center) and intracellular (right) views. Flow restrictor Tyr273 and Arg sandwich Arg92 and Arg258 residues are shown as sticks. PUFA site linoleic acid (EIC) is shown in space filling. Grey bars indicate membrane. Potassium ions are shown as purple spheres. b, Slice through the K_2P13.1(THIK-1) structure showing the locations of the intracellular vestibule, flow restrictor, pond, and selectivity filter (SF). c, K_2P13.1(THIK-1) extracellular view. M1, M2, M3, M4, P1, and P2 helices are labeled. SF residues Phe113 and Phe240 are shown as sticks. Other elements are displayed as in ‘a’. Cap is not shown. d, Details of the K_2P13.1(THIK-1) PUFA binding site. M1 (teal), M2 (magenta), P1 (salmon), and M4 (violet) helices are shown as cylinders. PUFA site residues are shown as sticks and are labeled. Flow restrictor Tyr273 is in italics. EIC is shown as sticks and transparent space filling atoms. e, Surface view of ‘d’. Select residues are labeled. EIC C8, C14, and C18 carbons are indicated. Colors are as in ‘d’. f, K_2P13.1(THIK-1) PUFA binding site hydrophilic interactions. Hydrogen bond network (black) surrounding the EIC acidic headgroup are shown. Arginine stack distance (grey) is indicated. g, and h, Structural superposition of K_2P13.1(THIK-1) (salmon and deep teal) and the K_2P2.1(TREK-1):ML335 complex (PDB:6CQ8) (yellow and light blue). g, Extracellular view showing position of the K_2P2.1(TREK-1) K_2P modulator pocket (orange oval). ML335 is shown as black sticks. EIC is shown as sticks and transparent space filling atoms. SF phenyalanine sidechains are shown as sticks. M1, M2, M3, M4, P1, and P2 helices are labeled. h, Side view showing overlap regions of K_2P modulator pocket and PUFA site (orange). ML335 upper (pink) and lower (blue) rings are indicated. EIC C8 and C14 carbons are indicated. K_2P2.1(TREK-1) residues are in italics. i, Closeup view of ‘h’

Figure 2. S136P mutation widens the K_2P13.1 (THIK-1) flow restrictor

a, Ribbon diagram superposition of the structures of K_2P13.1 (THIK-1) (salmon) and K_2P13.1 (THIK-1) S136P (cyan). Flow restrictor Tyr273 and Arg sandwich Arg92 and Arg258 residues are shown as sticks. PUFA site linoleic acid (EIC) is shown in space filling. Inset shows closeup view of flow restrictor. Cytoplasmic view is shown on the right. Potassium ions are purple spheres. b, Cryo-EM density (8σ) for flow restrictor regions of K_2P13.1 (THIK-1) (salmon) and K_2P13.1 (THIK-1) S136P (cyan). c, Cytoplasmic view of flow restrictor conformational changes. d, Slice through the K_2P13.1(THIK-1) S136P structure showing the locations of the intracellular vestibule, flow restrictor, pond, and selectivity filter (SF). K_2P13.1 (THIK-1) Tyr273 conformation (salmon) is shown as sticks. e, Structural comparison of S136P mutation site. Inset shows local conformation and hydrogen bond distances for K_2P13.1 (THIK-1) (salmon) and K_2P13.1 (THIK-1) S136P (cyan). Common distances are in black. f, Zoomed in view of S136P site. Figure is ~90° rotated relative to the view in the ‘a’ inset.

Figure 3. K_2P13.1 (THIK-1) flow restrictor controls inner cavity shape and function

a-c, Pore profile comparisons for a, K_2P13.1 (THIK-1) (deep salmon) b, K_2P13.1 (THIK-1) S136P (cyan), and c, K_2P2.1 (TREK-1) (PDB:6CQ6) (light blue) calculated using HOLE. Selectivity filter 2 (SF2), Flow restrictor, and Asn ring residues are shown as sticks. Select channel elements are labeled. d, K_2P13.1 (THIK-1) model and pore profiles. e, Representative TEVC current-voltage responses for K_2P13.1 (THIK-1) (black) and K_2P13.1 (THIK-1) mutants: S136P (cyan), I139A (purple), I136A/Y273A (orange), Y273A (magenta), Y273F (green), and Y273L (red). Inset shows protocol. f, Average currents I/I_<FL> at 0 mV for K_2P13.1 (THIK-1) (black) and K_2P13.1 (THIK-1) mutants: S136P (cyan), I139A (purple), I136A/Y273A (orange), Y273A (magenta), Y273F (green), and Y273L (red) where I_<FL> = average currents for full length K_2P13.1 (THIK-1). **** p <0.0001, *** p <0.001, ** p <0.01, * p = 0.01 –0.05, n.s. p >0.05. g, Pore profile of K_2P13.1 (THIK-1) S136P/Y273A (magenta). h, Potential of Mean Force (PMF) for K_2P13.1 (THIK-1) (black), K_2P13.1 (THIK-1) Y273F (green), K_2P13.1 (THIK-1) Y273A (magenta), K_2P13.1 (THIK-1) S136P (cyan), K_2P13.1 (THIK-1) S136P/Y273F (dashed green), and K_2P13.1 (THIK-1) S136P/Y273A (dashed dark magenta). Z axis corresponds to that shown in ‘d’. Statistical analysis was performed using the Kruskal-Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons test. In ‘d’ and ‘h’ locations of the Flow restrictor (light blue) and Selectivity filter (SF) (orange) are indicated.

Figure 4. K_2P13.1 (THIK-1) PUFA site is important for function

a, Representative TEVC current-voltage responses for K_2P13.1 (THIK-1) (black) and K_2P13.1 (THIK-1) mutants R92A (cyan), F101A (yellowgreen), Y102A (dark green), G105F (red), G105I (orange), S246A (green), R258A (maroon), N261A (blue), F262A (dark blue). Inset shows protocol. b, Average currents I/I_<FL> at 0 mV for K_2P13.1 (THIK-1) (black) and K_2P13.1 (THIK-1) mutants where I_<FL> = average currents for full length K_2P13.1 (THIK-1). Colors are as in ‘a’. Lipid head group coordinating and pocket lining residues are indicated. **** p <0.0001, *** p <0.001, ** p < 0.01, * p = 0.01 –0.05. Statistical analysis was performed using the Mann-Whitney test. c, Effects of mutants on K_2P13.1 (THIK-1) basal activity. Colors indicate increase (cyan), decrease (red), both (purple), or no effect. d-g, Representative TEVC current-voltage responses (d and f) and time course responses (e and g) for K_2P13.1 (THIK-1) (purple) and K_2P13.1 (THIK-1) R92A (red) to d-e, linolenic acid (EIC) and f-g, arachidonic acid (AA). d, and f, show lipid structure. Unsaturated bond locations are marked by numbers. Lipid class (ω6) is indicated. IV curves show baseline (black), lipid application (purple or red), and after 2’ washout (purple or orange dashed lines). h, Lipid modulation of K_2P13.1 (THIK-1) and K_2P13.1 (THIK-1) R92A I/I₀ at 0 mV where I₀ is the current before lipid application. Light purple and orange show response after 2’ washout. Lipids are: arachidonic acid (AA), arachidonic acid methyl ester (AAMe), eicosapentanoic acid (EPA), dihomo-γ-linolenic acid (DGLA), α-linolenic acid (α-LNL), linoleic acid (EIC), oleic acid (OA), and stearic acid (SA). X:Y indicates number of carbons and unsaturated bonds. **** p <0.0001, *** p <0.001, ** p < 0.01, * p = 0.01 –0.05, n.s. p >0.05. Statistical analysis was performed using the Kruskal-Wallis test (nonparametric ANOVA) followed by Dunn's multiple comparisons test. i, TEVC dose response curve for K_2P13.1 (THIK-1) R92A activation by linoleic acid. Curve shows fit to the Hill Equation. EC₅₀= 0.7 µM, Hill coefficient ‘n’ = 2.0. j, Model of THIK function. Changes in the conformation equilibrium affect ion passage. k, Comparison of K_2P13.1 (THIK-1) (deep salmon) and Kv7.1 (KCNQ1) (marine and light blue) (PDB:6UZZ) P1/M4 and P/S6 interfaces. Residues framing the putative Kv7.1 (KCNQ1) PUFA site, Lys326 and Asp301 and EIC (yellow orange) are shown in space filling. Arginine sandwich pair is indicated.