Multiple modalities converge on a common gate to control K2P channel function

Abstract

Members of the K(2P) potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K(2P)s are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K(2P), K(2P)2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K(2P)s and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K(2P) family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.

Figure 1

Functional selection identifies GOF mutations in a mammalian K_2P2.1 (TREK-1). (A) Growth of potassium-transport-deficient yeast (SGY1528) expressing the yeast potassium transporter TRK1, Kir2.1, an inactive Kir2.1 mutant (Kir2.1^*), K_2P2.1 (TREK-1), and two exemplar K_2P2.1 (TREK-1) GOF mutants, L267P and W275S under non-selective conditions (100 mM KCl), and two different selective conditions (1 mM and 0.5 mM KCl). Rows indicated with ‘Ba²⁺’ show growth in the presence of the Kir2.1 and K_2P2.1 (TREK-1) inhibitor 8 mM BaCl₂. (B) K_2P2.1 (TREK-1) subunit topology diagram. Locations of GOF mutations are indicated in yellow. Transmembrane segments M1, M2, M3, and M4 and the two P-loop domains are labelled. (C) Exemplar current–voltage traces from whole cell recordings of Xenopus oocytes injected with 0.3 ng of K_2P2.1 (TREK-1) or GOF mutant mRNA. Currents were elicited in solutions containing 2 mM potassium (ND96) by a ramp protocol from −150 to +50 mV from a −80 mV holding potential. Values for the average current (in μA, mean±s.e., n=5) at 0 mV were K_2P2.1 (TREK-1) (0.52±0.40), I148T (1.61±0.106), L267P (1.77±0.121), I148T/L267P (3.11±0.092), W275S (4.86±0.099), F276L (1.70±0.097). (D) Quantification of normalized current amplitudes at 0 mV from Xenopus oocytes injected with 0.3 ng of mRNA for the indicated channels. (E) Cell-attached mode single channel recordings of K_2P2.1 (TREK-1), I148T/L267P, and W275S expressed in COS7 cells. O₁, O₂, and O₃, indicate the first, second, and third open states, respectively. C indicates the closed state. (F) Open channel probabilities from single channel analyses calculated on recordings of ∼30 s duration. K_2P2.1 (TREK-1) (n=7), I148T (n=7), L267P (n=5), I148T/L267P (n=9), W275S (n=9), F276L (n=8). Data represent mean±s.e.

Figure 2

K_2P2.1 (TREK-1) GOF mutations affect response to extracellular acidosis, heat, and pressure. (A) Exemplar two-electrode voltage-clamp recordings of the response of K_2P2.1 (TREK-1) and the W275S GOF mutant to external pH (pH_O) changes in 2 mM [K⁺]_O solutions. (B) Normalized pH_O responses (at 0 mV) for the indicated channels. (C) Exemplar two-electrode voltage-clamp recordings of the response of K_2P2.1 (TREK-1) and the W275S GOF mutant to temperature in 2 mM [K⁺]_O, pH 7.4 solutions. (D) Normalized temperature responses (at 0 mV) for the indicated channels. (E) Exemplar mechanical force (cell-attached mode, 150 mM KCl pH 7.2 in the bath, 5 mM KCl pH 7.4 in the pipette) responses of K_2P2.1 (TREK-1) and the I148T/L267P GOF mutant stimulated by negative pressure applied to the extracellular side of the plasma membrane through the patch pipette. (F) Normalized pressure responses for the indicated channels. In (A, C) currents were elicited by a ramp from −150 to +50 mV from a holding potential of −80 mV. Lines for (B, D) show fits to the equations I=I_min+(I_max–I_min)/(1+([H⁺]_O/K_1/2)^H) and I=I_min+(I_max–I_min)/(1+e_1/2^(T−T)/S), respectively. Data in (B, D, F) show mean±s.e. (n=8–30). N⩾2 for all experiments.

Figure 3

Extracellular loops and extracellular proximal portion of M4 control K_2P2.1 (TREK-1) gating. (A–C) Normalized response to pH_O in low (2 mM, data are from Figure 2B) and high (90 mM) [K⁺]_O (2 K and 90 K, respectively) for K_2P2.1 (TREK-1) and the indicated GOF mutants. Whole cell currents were elicited in Xenopus oocytes by a ramp from −150 to +50 mV from a holding potential of −80 mV (2 K) or 0 mV (90 K). (D) Quantification of the effect of high (90 mM) external potassium on the pH_O response from the curves in (A–C) I_6.5norm(90 K)/I_6.5norm(2 K). Lines for (A–C) show fits to the Hill equation ((I=I_min+(I_max–I_min)/(1+([H⁺]_O/K_1/2)^H)). Data represent mean±s.e. (n=6–30). Statistical analysis: t-test. ^***P<0.001, NS, not significant (P>0.05). N⩾2 for all experiments.

Figure 4

Extracellular loops and extracellular proximal portion of M4 control ion selectivity of K_2P2.1 (TREK-1). (A–D) Exemplar two-electrode voltage-clamp recordings of the response of the wild-type and mutant K_2P2.1 (TREK-1) channel to pH_O changes in 100 mM external sodium solutions. Currents were evoked by 60 ms long pulses from −150 to −50 mV in 10 mV increments from a −80 mV holding potential. Cells were injected with different amounts of mRNA to yield comparable current amplitudes. (E) Exemplar two-electrode voltage-clamp recordings of the response of the wild-type K_2P2.1(TREK-1) channel to pH_O changes in 100 mM external potassium solutions. Currents were evoked by 60 ms long pulses from −50 to 60 mV in 10 mV increments from a 0-mV holding potential. (F) Quantification (mean±s.e., n=8–11) of apparent permeability ratios at different pH_O using the equation pNa/pK=e^FΔErev/RT, where pNa and pK are permeabilities for sodium and potassium, respectively, and ΔErev is a difference between the reversal potentials measured in 100 mM sodium and 100 mM potassium solutions. N⩾2 for all experiments.

Figure 5

Tests of the impact of amino-acid changes at Trp275 on K_2P2.1 (TREK-1) function. (A) Normalized whole cell current amplitude (at 0 mV) recorded in 2 mM [K⁺]_O pH 7.4 from Xenopus oocytes injected with equivalent amounts of mRNA for the indicated K_2P2.1 (TREK-1) Trp275 substitutions. Statistical analysis: t-test. ^***P<0.001; NS, not significant (P>0.05). (B) Normalized pH_O responses in 2 mM [K⁺]_O for the indicated Trp275 mutants. (C) Normalized temperature responses in 2 mM [K⁺]_O for the indicated W275 mutants. Curves show fits to the Hill equation I=I_min+(I_max–I_min)/(1+([H⁺]_O/K_1/2)^H) or I=I_min+(I_max–I_min)/(1+e_1/2^(T−T)/S). Data represent mean±s.e. (n=4–30). N⩾2 for all experiments.

Figure 6

Importance of the Trp275 position is functionally conserved among K_2P channels for both pH_O and temperature induced gating. (A) Amino-acid alignment of the M4 region from the indicated K_2P channels. Residues conserved in three or more of the indicated sequences are highlighted in blue. The K_2P2.1 (TREK-1) Trp275 homologous position is indicated in orange. (B–E) Normalized pH_O responses and exemplar two-electrode voltage-clamp recordings of Xenopus oocytes for the indicated channels and mutants (2 mM [K⁺]_O). Injected mRNA amounts for the exemplar traces in ng are as follows for wild type and mutant, respectively: K_2P10.1 (TREK-2) 5.0, 1.0; K_2P9.1 (TASK-3) 0.08, 0.20; K_2P3.1 (TASK-1) 1.5, 5.0; K_2P5.1 (TASK-2) 2.0, 1.0. (F–I) Normalized temperature responses for the indicated channels and mutants (2 mM [K⁺]_O, pH 7.4). Exemplar two-electrode voltage-clamp records are shown only for K_2P10.1 (TREK-2). Data represent mean±s.e. (n=6–15) and is fitted to I=I_min+(I_max–I_min)/(1+([H⁺]_O/K_1/2)^H) or I=I_min+(I_max–I_min)/(1+e_1/2^(T−T)/S). Dashed lines connect data points. N⩾2 for all experiments.

Figure 7

Temperature gating responds to changes in external potassium concentration. Normalized temperature responses of (A) K_2P2.1 (TREK-1), K_2P2.1 (TREK-1) W275S, and K_2P2.1 (TREK-1) F276L and (B) K_2P10.1 (TREK-2) from two-electrode voltage-clamp recordings in 2 and 90 mM [K⁺]_O (2 K and 90 K, respectively) elicited by a ramp from −150 to +50 mV from a holding potential of −80 mV (2 K) or 0 mV (90 K) at indicated temperatures at pH 7.4. The K_2P2.1 and K_2P10.1 responses to heat in 2 mM [K⁺]_O are the same as in Figures 2D and 6F, respectively. Data (mean±s.e., n=8–15) were taken at 0 mV (2 K) or +40 mV (90 K) and fitted with the equation I=I_min+(I_max–I_min)/(1+e_1/2^(T−T)/S). N⩾2 for all experiments.

Figure 8

Cartoon model of K_2P2.1 (TREK-1) C-type-like gating by extracellular acidosis, heat, and pressure. Cartoon model of how extracellular protons ([H⁺]_O), heat, and pressure affect the transition of K_2P2.1 (TREK-1) between a low-activity (inhibited) state and a high-activity (activated) state that involves a C-type-like gate. As suggested earlier (Sandoz et al, 2009), external acidification causes structural rearrangements in the pore triggered by electrostatic interactions between the protonated extracellular pH-sensing His126, and the negatively charged region in the P2-loop, which includes Asp263 and Glu265. We propose that Ile148, Leu267, and Trp275 (indicated with stars) are crucial elements of the C-type-like gate. As suggested elsewhere (Honore, 2007), temperature and mechanical stress have their sensing elements located in the intracellular C-terminal domain. We further propose that activated C-terminal domain (indicated with the orange halo) induces movements of the M4 transmembrane segment and affects channel activity through the C-type gate. The blue arrow indicates the putative pathway. For clarity, only one of the two cytoplasmic C-termini is depicted.