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Fig. 1. State-dependent modification of inner pore cysteine residues in TALK-2 K2P channels.a Representative current trace measured at +40 mV from an inside-out patch expressing WT TALK-2 channels with symmetrical K+ concentrations at pH 7.4. Channel currents were activated with the indicated compounds (1.0 mM 2-APB and 5.0 µM oleoyl-CoA) applied to the intracellular membrane side. Inlays show current-voltage responses of 2-APB- (blue) and oleoyl-CoA-activated (green) channels compared to basal state (black) using the indicated voltage step protocol. b Pore homology model of TALK-2 based on the crystal structure of TASK-1 (PDB ID: 6RV3, chains A, B) with the SF highlighted red, K+ ions black, and introduced cysteine residues (L145C and Q266C) for MTS-ET modification yellow. c Pore cavity zoom-in displaying the localization of L145C in the inner cavity and Q266C at the intracellular end of the pore. d Representative measurement of TALK-2 L145C channels showing state-dependent MTS-ET modification with no effect under unstimulated (basal) conditions or inhibition upon application of 1.0 mM MTS-ET in pre-activated states with 1.0 mM 2-APB (blue) or 5.0 µM oleoyl-CoA (green) with the indicated time constants (τ), respectively. e Measurement as in (d) with TALK-2 Q266C channels showing state-independent modification with activation upon application of 1.0 mM MTS-ET. f Current responses recorded using the indicated voltage step protocol in symmetrical K+ showing activation of WT TALK-2 with increasing 2-APB concentrations. The dotted line shows the increase and saturation of current amplitudes with 2-APB at + 40 mV. g 2-APB dose-response curves analyzed from measurements as in f for WT TALK-2 (blue, n = 26), TALK-2 L145C (black, n = 7), and WT TREK-1 (gray, n = 28) channels. h Correlation between the fold change in current amplitudes of TALK-2 L145C channels at +40 mV (black squares) and the rate of MTS-ET modification (1/τ) at +40 mV (orange squares) with different 2-APB concentrations. Data shown are the mean ± s.e.m and the number (n) of independent experiments is indicated in the figure and supplementary tables 1 and 2. The representative experiments were repeated with the similar results as indicated in the figure.
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Fig. 2. The lower constriction functions as a permeation gate.a Representative measurement of WT TALK-2 channels from an inside-out patch in symmetrical K+ at +40 mV with increasing 2-APB concentrations (c1–c5) applied from the intracellular side at indicated time points (blue arrows). At steady-state, current levels with 2-APB intracellular K+ was exchanged by Rb+ showing an enhanced activatory Rb+ ion effect on the SF in 2-APB pre-activated channels. b Recording as in a for WT TREK-1 K2P channels showing the stepwise loss of Rb+ activation in the presence of increasing 2-APB concentrations. c Correlation of Rb+-induced currents from measurements as in a, b in the presence of 0.01, 0.1, 0.5, 1.0, or 2.0 mM 2-APB for either WT TALK-2 (black, n = 12) or WT TREK-1 (gray, n = 5) channels. d Gating scheme highlighting the effect of 2-APB and Rb+ on the lower and selectivity filter gate in TALK-2 channels. Data shown are the mean ± s.e.m and the number (n) of repeats of the representative measurements with similar results is indicated in the figure.
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Fig. 3. Functional characterization of the lower gate in TALK-2 K2P channels.a Relative current amplitudes from TEVC measurements at pH 8.5 of WT and mutant TALK-2 channels. Currents were elucidated with a voltage protocol ramped from -120 mV to +45 mV within 3.5 s, analyzed at +40 mV and normalized to WT. Inlays showing representative WT TALK-2 (gray traces), TALK-2 L264A, L262A, W255A and V146A mutant channel currents (blue and green traces), respectively and a topology model of a channel protomer highlighting the localization of the g-o-f mutations. b Sequence alignment of the TM2, TM2-TM3 linker, and TM4 regions of the human K2P channels TALK-2 and TASK-1. c Pore homology model of TALK-2 based on the crystal structure of TASK-1 (PDB ID: 6RV3, chains A, B) highlighting the cluster of g-o-f mutations (V146A, L262A and L264A) at the cytosolic pore entrance. d Representative inside-out single channel measurements of WT TALK-2 (gray trace) and TALK-2 L264A mutant channels (blue trace) at -100 mV. e Relative open probability (NPO) and single channel amplitudes (SCA) analyzed from recordings as in d for WT and L264A TALK-2 channels (n = 6). f–h Analysis of the mean channel-open times (g), closed time events (f), and burst behavior (h; see “Methods” section) for WT and TALK-2 L264A mutant channels. i Representative measurement of TALK-2 L264A mutant channels additionally carrying the inner pore mutation L145C (TALK-2 L264A/L145C) at +40 mV showing a fast and irreversible modification and subsequent block upon application of 1.0 mM MTS-ET. The experiment was repeated with similar results (n = 10). j Cartoon illustrating the pore accessibility of MTS-ET in L145C mutant TALK-2 channels with or without carrying an additional g-o-f mutation. k Modification rates of WT, L145C and double mutant TALK-2 channels at +40 mV as indicated. Data shown are the mean ± s.e.m and the number (n) of independent experiments is indicated in the figure and supplementary table 2. Statistical relevance has been evaluated using unpaired, two-sided t-test and exact P values are indicated in the figure. n.d. not determinable.
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Fig. 4. Direct stimulation of the SF produces a state-dependent cysteine modification in the pore of TALK-2.a TALK-2 current responses to voltage step families as indicated using symmetrical K+ concentrations (120 mM [K+]ex./120 mM [K+]int.) at pH 7.4 on both sides (black traces), or at extracellular pH 9.5 (brown traces). b Cartoon illustrating a simplified TALK-2 channel gating model and pore accessibility to MTS-ET by alterations of the pHe that directly affects the SF. c Representative modification and subsequent irreversible inhibition with 1.0 mM MTS-ET of TALK-2 L145C channels pre-activated by extracellular alkalinization (pHe 9.5). d TALK-2 channel currents with intracellular Rb+ (120 mM [K+]ex./120 mM [Rb+]int.) at pH 7.4 for different potentials as indicated showing a maximum PO reached for potentials positive to ~+135 mV (Vmax), as further depolarizations do not increase the tail current amplitudes. e Voltage activation (conductance-voltage (G–V) curves) with V1/2 values of 72 ± 2 mV and 66 ± 3 mV of WT TALK-2 (n = 15) and L145C mutant channels (n = 10), respectively. The highlighted voltages (orange) represent the voltage activation levels for MTS-ET modification experiments shown in i. f Cartoon of a simplified gating model with Rb+ as an amplifier for voltage activation targeting the SF and subsequently the lower gate in TALK-2 channels. g, h Representative measurements at +40 mV of WT (g) and L145C mutant TALK-2 channels (h) showing a non-modifiable state or an almost complete modification/inhibition with 1.0 mM MTS-ET within 60 s in intracellular Rb+, respectively. i Correlation between the fold change of tail current amplitudes (black squares) of TALK-2 L145C channels and the incidental rate of MTS-ET modification (1/τ) (orange squares) with intracellular Rb+ at different potentials as indicated. Data shown are the mean ± s.e.m and the number (n) of independent experiments and repeats of representative measurements with similar results is indicated in the figure and supplementary tables 1–3.
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Fig. 5. Open channel blocker show state-dependent pore accessibility and slowing of deactivation kinetics in TALK-2.a Current responses of WT TALK-2 channels activated with indicated voltage steps under symmetrical ion conditions with either intracellular K+ (black trace, basal state) or Rb+ (red trace, activated state) and with 1.0 mM TPenA in Rb+ (orange trace). Note, the presence of TPenA shows slowing of deactivation resulting in a tail current cross-over. Cartoon depicting a simple model for TALK-2 channel gating, whereby Rb+ activation of the SF enables blocker (e.g., TPenA) binding in the pore and unbinding facilitates lower and SF gate closure at −80 mV. b Same recording as in (a) with TREK-2 channels showing inhibition with 50 µM TPenA without tail current cross-over. c Representative current responses of WT TALK-2 channels to voltage steps as indicated in the absence (black) and presence of 1.0 mM TPenA (orang) applied to the intracellular membrane side. d Representative measurement of TALK-2 channel currents at +40 mV showing dose-dependent TPenA inhibition in the pre-activated state with 1.0 mM 2-APB. e Dose-response curves of TPenA inhibition from measurements as in d for TALK-2 in unstimulated conditions (black, n = 16) and pre-activated states with 2-APB (blue) with altering apparent affinities for TPenA (IC50 (0.2 mM 2-APB, n = 7) = 778 ± 116, IC50 (0.5 mM 2-APB, n = 5) = 215 ± 28, IC50 (1.0 mM 2-APB, n = 16) = 54 ± 10). f Residual currents of WT and L264A mutant TALK-2 channels at +40 mV upon 1.0 mM TPenA block at indicated conditions. g Residual currents of unstimulated (black), 2-APB pre-activated WT (blue) and L264A mutant (gray) TALK-2 channels after inhibition with the indicated blocker. h Simplified gating scheme indicating that blocker interact with the open state of TALK-2 to produce inhibition. Data shown are the mean ± s.e.m and the number (n) of independent experiments and repeats of representative measurements with similar results is indicated in the figure. Statistical relevance has been evaluated using unpaired, two-sided t-test and exact P values are indicated in the figure.
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Fig. 6. Impact of ligand modulation on SF energetics in TALK-2 K2P channels.a–c
G–V curves analyzed from current-voltage families (−120 mV to +160 mV with 5 mV increments) measured under symmetrical ion conditions with intracellular Rb+ of WT TALK-2 (a), WT TREK-2 (b) and L264A mutant TALK-2 channels (c) in the absence (black traces, n = 15 TALK-2, n = 6 TREK-2, n = 15 TALK-2 L264A) and presence of 0.1 mM (brown trace, n = 7 TREK-2) or 1.0 mM TPenA (orange traces, n = 8 TALK-2, n = 7 TREK-2, n = 8 TALK-2 L264A), respectively. d
G–V curves analyzed from WT TALK-2 tail currents in the presence of pHe 7.4 (black trace, n = 15), pH 9.0 (blue trace, n = 6), and pH 10.5 (green trace, n = 6). e V1/2 values from G–V curves analyzed as in d with varying pHe (pHe 7.0, n = 6; pHe 7.4, n = 15; pHe 8.0, n = 6; pHe 8.5, n = 7; pHe 9.0, n = 6; pHe 9.5, n = 9; pHe 10.0, n = 9; pHe 10.5, n = 6). f V1/2 values from G–V curves of WT TALK-2 channels activated with 1.0 mM 2-APB (n = 5), 5.0 µM oleoyl-CoA (n = 7) or inhibited with 1.0 mM TPenA (n = 8). Dashed lines in e, f represent the level of WT (unstimulated) V1/2 at pHe 7.4. g Normalized currents from TEVC measurements of oocytes expressing WT (n = 11) and mutant L262A (n = 5) or L264A TALK-2 channels (n = 8), respectively. Channels were activated by increasing pHe from 5.5 to 10.5 with 0.5 pH increments. Currents were elucidated with a voltage protocol ramped from −120 mV to +45 mV within 3.5 s, analyzed at +40 mV and normalized to pH 10.5. Data shown are the mean ± s.e.m and the number (n) of independent experiments is indicated in the figure and supplementary tables 3 and 4.
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Fig. 7. Functional coupling of the SF and the lower gate in TALK-2 K2P channels.a, b
G–V curves analyzed from tail currents at -80 mV after 300 ms pre-pulse steps (-120 mV to +160 mV with 5 mV increments) under symmetrical ion conditions with intracellular Rb+ of WT (n = 15) and V146A (n = 6), W255A (n = 14), L262A (n = 8), and L264A mutant TALK-2 channels (n = 15), respectively (b) and the summary of V1/2 values from Boltzmann fits to the corresponding G–V curves (a). Dashed line in a represents the level of WT (unstimulated) V1/2 at pHe 7.4. c Correlation of the V1/2 shifts of mutant TALK-2 channels at basal and WT TALK-2 channels at indicated conditions with the time constants of modification of TALK-2 L145C channels under the corresponding activatory conditions or in combination with the respective g-o-f mutation. d Simplified energetic scheme depicting the electrical work (∆G = zF∆V1/2) required to open both gates (∆Gtotal) with the individual contribution of the SF gate (∆GSF) and lower gate (∆GLG). Mutations (as indicated in the inlay) that open the lower gate reduced this electrical work as seen in the positive V1/2 shifts of the G–V curve. Note, our results actually show that both gates are strongly positively coupled and, thus, the pre-open state (with only the lower gate open) is just a conceptual state to illustrate the energetic contribution of the lower gate. Data shown are the mean ± s.e.m and the number (n) of independent experiments is indicated in the figure and supplementary tables 3 and 4. n.d. not determinable, n.e. no expression.
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