Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel

Abstract

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.

Figure 1. The cytosolic proximal C-terminus controls the pore opening induced by 2-APB without depending the conformation of selectivity filter.

(A) Cartoon diagram of a single TREK-2 subunit, transmembrane segments 1 ~ 4 (M1 ~ M4), intracellular N-terminus, C-terminus (Ct), proximal C-terminus (pCt) and key residues are indicated. (B) Exemplar current-voltage recordings from oocytes expressing TREK-2 or ΔpCt in the presence of 100 μM 2-APB. (C) Exemplar current-time recordings from oocytes expressing the WT TREK-2 channels and ΔpCt deletants in the presence of 100 μM 2-APB. (D) Comparative analysis of the dose-response curves evoked by 2-APB between TREK-2 and ΔpCt. Due to the lower sensitivity of ΔpCt to 2-APB, higher concentrations were used in the curve. Only two points (100 μM and 200 μM) were tested for statistical significant analysis. (E,F) Comparative analysis of the concentration-response curves of TREK-2 channels in the absence and presence of 5 mM, 20 mM extracellular K⁺ (E), and the indicated S4 (K⁺ binding site 4) mutants (F) activated by 2-APB.

Figure 2. Functional characterization of the Gly312 hinge in the 2-APB response of TREK-2 channels.

(A) Sequence alignment of the M4 region of indicated channels. The locations of Glycine hinge are indicated. (B) Exemplar current-time recordings from oocytes expressing the WT TREK-2 channels and G312A in the presence of 100  μM 2-APB. (C) Comparative analysis of the dose-response curves evoked by 2-APB between TREK-2 and G312A.

Figure 3. The cytosolic proximal C-terminus is involved in the gating process of selectivity filter induced by extracellular alkalization.

(A) Exemplar current-voltage recordings from oocytes expressing TREK-2 or ΔpCt as pH_o transitions among 6.5, 8.0 and 9.3. (B) Normalized responses of indicated channels to pH_o changes. The currents were normalized by the ones recorded at pH6.5 (I_pH6.5).

Figure 4. Functional characterization of the Gly312 hinge in the pHo response of TREK-2 channels.

(A) Exemplar current-voltage recordings from oocytes expressing the WT TREK-2 channels and G312A as pH_o transitions among 6.5, 8.0 and 9.3. (B) Concentration-dependence for extracellular alkalization inhibition of TREK-2 and G312A.

Figure 5. Functional characterization of the Phe316 in the 2-APB and pH_o response of TREK-2 channels.

(A) Sequence alignment between TREK-2 M4 and KcsA M2. The location of Phe316 is indicated. (B) Comparative analysis of the dose-response curves evoked by 2-APB between TREK-2 and G312A. (C) Normalized responses of indicated channels to pH_o changes.

Figure 6. Delineation the roles of Glycine hinges in TREK-1 channels.

(A) Cartoon diagram of a single TREK-1 subunit, transmembrane segments 1 ~ 4 (M1 ~ M4), the intracellular N-terminus, C-terminus (Ct), proximal C-terminus (pCt) and the key residues are indicated. (B) Quantification of the effect of 2-APB on the indicated channels. (C–E) Comparative analysis of pH_o response curves for the indicated channels.