K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac

Abstract

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Fig. 1. Crystal structures of two distinct conformations of TREK-2.

TREK-2 overall fold viewed parallel to the plane of the membrane, with views of (A) the up state, 3.4 Å structure; (B) the down state, 3.9 Å structure and (C) a superposition of the states and (D) a view of the superposition from the cytoplasmic side of the membrane. The up state chains A and B are colored yellow and purple, the down state chains A and B are colored orange and blue. Potassium ions are colored green and the disulfide bond in the cap is shown in red. (E) View of the lateral fenestration (indicated with an arrow) in the down state, showing the aliphatic chain which binds in the vestibule and the fenestration, and lipids (Lip1, Lip2, Lip3) bound to the structure (green sticks). (F) View of the same region as in E in the up state, showing the absence of the fenestration when the helices are in the up state, with Phe³¹⁶ and Leu³²⁰ blocking the fenestration. Hinge point around G³¹² in M4 is indicated by an open triangle.

Fig. 2. Norfluoxetine and Br-Fluoxetine bind to TREK-2 in the fenestration adjacent to the pore filter entrance.

(A) The overall fold of TREK-2 down state is shown with the 5 Å anomalous difference map for the Br-fluoxetine/TREK-2 complex shown in pink (contoured at 4.5 sigma), indicating the location of the Br-fluoxetine in the fenestration. (B) The chemical structure of Br-fluoxetine is shown. Norfluoxetine, the active metabolite of fluoxetine, lacks the methyl group attached to the nitrogen atom. (C) Cross section of a surface view of TREK-2 in the down state, with the surface colored according to hydrophobicity (color ramped from green for most hydrophobic, through yellow to blue for least hydrophobic). (D) The complex of TREK-2 with norfluoxetine. Norfluoxetine is shown in cyan, blue, red and orange for carbon, nitrogen, oxygen and fluorine atoms. For clarity, only one enantiomer of norfluoxetine is shown in C/D. (E) The norfluoxetine binding site, with chain A shown in gold and chain B shown in blue, norfluoxetine is colored as in D. (F) Disruption of the binding site by the L320W mutation reduces norfluoxetine inhibition. (G) Stretch activation (-11 mmHg, red) at internal pH 7.3 dramatically reduces the efficacy of norfluoxetine inhibition, as does activation by 10 µM arachidonic acid (AA, blue).

Fig. 3. Concerted motion of transmembrane helices (M2, M3, M4).

(A) Superposition of the up and down states highlighting motion in M2-M4 transmembrane helices. (B) Interactions between helices M2/M3/M4 in the down state, showing the interactions between Trp³²⁶, Arg²³⁷ and Met³²². View shown is indicated by black boxed region in A. The lipid chain that overlays this interface is shown in green. (C) View of corresponding region in the up state, showing disruption of distal M3/M4 interface due to shift in M4 orientation. (D-E) Interactions at the top of the M2/M3/M4 helices, proximal to the pore in the down (D) and up (E) states viewed from direction indicated by blue boxed region in A. TM helix hinge points are indicated by grey spheres. (F) Mutations at the interface between M2, M3 and M4 reduce the fold-activation by membrane stretch (-11 mmHg).

Fig. 4. The external pH sensor region centered on His¹⁵⁶.

(A) The sensor histidine (His¹⁵⁶, green) is located between the ECD (pale blue) and pore helix 1 (PH1) of the transmembrane domain adjacent to the pore region (B-C). The environment round His¹⁵⁶ is shown with different structural elements colored. Dotted lines represent hydrogen bonds. The filter loops P1-M2 and P2-M4 are colored orange and yellow respectively. (D-E) Effects of mutating residues within either the His¹⁵⁶ network (D) or the internal hydrophobic hinge between M2-M4 (E) on external pH-sensitivity.

Fig. 5. Model of K2P channel gating and inhibition by norfluoxetine.

The down state, lower conductivity conformations are shown in orange, with the fenestrations indicated in yellow. The up state conformations are shown in blue. Inhibitors such as norfluoxetine are represented by a red triangle. (A) Overall scheme for K2P channel gating. Higher and lower conductivity states are shown for both conformations as the filter may gate independently of these larger changes, though with a different probability. Conformations for which we have crystal structures are indicated with an asterisk. (B) Schematic of activation of TREK channels by mechanical stretch. The direct interaction of TREK-2 with lipids in the membrane may allow lateral forces to facilitate conversion from the down to the up state which is fluoxetine insensitive. (C) Association of the C-terminal domain (CTD) with the membrane. The diverse cytoplasmic C-terminal extensions of K2P channels provide an additional site for modulation of channel activity. This regulation could operate in part though association of the CTD with the membrane after post-translational or pH changes that would favor the more active up state.