Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the selectivity filter gate

Figures

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  Figure 1.. The current model for TREK channel gating and NFx-binding sites. (A) The TM helices exist in two states (up and down) but it is unclear whether opening of the filter gate requires movement to the up conformation (via route a), whether it can open independently in the down state (via route b), or even whether both options are possible. Current models also suggest that openings from the down state may result in a lower-activity channel than when it is in the up state, because many activatory mechanisms (e.g., membrane stretch) promote movement to the up state. Binding sites for NFx do not exist in the up state, and NFx binding will alter the equilibrium between these two conformations of the TM helices, but is unclear whether NFx binding is state dependent and only stabilizes the closed state of the channel. The presence of positively charged NFx bound within the inner pore may also cause direct pore block and/or allosteric effects on the filter gating mechanism itself. (B) Left: A view of the structure of TREK-2 in the down state showing NFx (as vdW spheres) bound within the fenestrations (PDB accession no. 4XDK. K+ ions in the filter are shown as purple spheres. Right: Expanded…

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  Figure 2.. Direct and allosteric interactions of NFx with TREK-2. (A) Representative traces of macroscopic TREK-2 currents elicited by voltage ramps between −80 and +80 mV in giant excised patches from Xenopus oocytes measured in control solution, in the presence of 50 µM ML335 alone, and with various added bath concentrations of NFx, as indicated. (B) Similar representative traces of TREK-2 currents in the absence (control) or presence of the activator, 1 mM 2-APB alone, and with added concentrations of NFx, as indicated. (C) Dose–response curves for NFx inhibition of TREK-2 currents on their own (IC50 = 2.7 µM; h = 1.7, n = 19) or in the presence of 1 mM 2-APB (IC50 = 3.8 µM; h = 0.6, n = 11) or 50 µM ML335 (IC50 = 164 µM; h = 0.8, n = 7), as indicated. Note the large shift in NFx sensitivity that results from ML335 activation, but not 2-APB activation. (D) Modified gating cartoon indicating gating modes with different activities rather than distinct open/closed states. The red arrow shows that 2-APB promotes a highly active state with unaltered NFx sensitivity, suggesting NFx inhibition is not state dependent. Other factors such as membrane stretch promote formation of various…

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  Figure S1.. Two types of TREK-2 behavior in lipid bilayers. (A and B) Single-channel recordings of TREK-2 incorporated into a bilayer at +80mV (top trace) and −80 mV (bottom trace). The dotted line represents the closed-channel level. (C and D) Macroscopic current–voltage relationships simulated for 100 TREK-2 channels using values of single-channel open probability (PO) and single-channel current amplitude (i) obtained from single-channel recordings of TREK-2 with standard (C) and high-PO behavior (D). The lines are fit by hand.

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  Figure S2.. Comparison of single-channel kinetics of TREK channels in standard and high-PO mode. (A) Outward currents. Top traces: Single-channel recordings of TREK-2 in the standard (top trace, PO = 0.14) and high-PO mode (bottom trace, PO = 0.92) at +60 mV. Dotted line represents the closed-channel level. Bottom panels: Distributions of single-channel openings (left) and closures (right) obtained from recordings of TREK-2 in standard and high-PO mode at +60 mV. (B) Inward currents. Top traces: Single-channel recordings of TREK-2 in standard (top trace, PO = 0.013) and high-PO mode (bottom trace, PO = 0.70) at −60 mV. Dotted line represents the closed-channel level. Bottom panels: Distributions of single-channel openings (left) and closures (right) obtained from recordings of TREK-2 in standard and high-PO mode at −60 mV. The number of exponential components in the dwell-time distributions in both A and B was determined by the least-squares method.

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  Figure S3.. Effects of NFx on the physical properties of the membrane. The relationship between the modulus of elasticity in the perpendicular direction of the membrane in the presence of NFx (E⊥NFX), normalized to that in control solution (E⊥(0)). The number of experimental values is shown above each point. The line is fit by hand.

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  Figure 3.. Effect of 100 µM NFx on a single TREK-2 channel. Single-channel recordings of TREK-2 in in the high-Po gating mode at different membrane voltages between +100 mV and −100 mV in the absence (left) and presence (right) of 100 µM NFx. Dotted lines represent the closed-channel levels.

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  Figure 4.. Summary of the effects of NFx on TREK-2 single channels. (A) The mean single-channel conductance of TREK-2 in the absence (open circles; n = 3) and presence of 100 µM NFx (filled circles; n = 3). (B) Histograms of open single-channel current level in the absence (black line) and presence (gray line) of 100 µM NFx at +100 mV (top) and −100 mV (bottom). (C) Mean single-channel open probability of TREK-2 in the absence (open circles; n = 3) and presence of 100 µM NFx (filled circles; n = 3). (D) Mean single-channel current conductance (filled squares) and mean single-channel open probability (filled circles) in the presence of 100 µM NFx normalized to values obtained in the absence of NFx (n = 3).

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  Figure S4.. Voltage dependence of NFx block at depolarized potentials. Dose–response relationships were determined at different voltages for NFx inhibition of macroscopic currents in giant excised patches from oocytes expressing WT TREK-2. At saturating concentrations, relatively little voltage dependence is observed, but at depolarized potentials, a small shift is observed. The lines are fit with a Hill inhibition equation (Eq. 1) assuming a = 0. The IC50 values are 4.2 µM, h = 0.72 (0 mV); 2.0 µM, h = 0.74 (+40 mV); and 1.4 µM, h = 0.79 (+80 mV).

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  Figure S5.. Single-channel properties in the presence of NFx. (A and B) Relative Po does not define the efficacy of NFx inhibition. Single-channel recordings at +60 mV of a single TREK-2 channel reconstituted in a bilayer with low (A) and high Po (B) in the absence and presence of 10 µM NFx as indicated. Dotted line represents the closed-channel level. The relative change in Po is similar in both cases. (C–F) Properties of long closed states and bursts in the presence of NFx. Mean lifetimes (C) and relative areas (D) of three apparent long closed states observed in the presence of 100 µM NFx in single-channel recordings depicted in Fig. 3. The lines through the data are fit by hand. (E and F) The dependence of the mean burst duration and the mean interburst closure on the membrane voltage in the presence of 100 µM NFx. This shows that the increase in NFx inhibition above +60 mV is accompanied by both a decrease in the mean burst length and an increase in the mean interburst close time. The lines are fit by hand.

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  Figure 5.. Effect of NFx on single-channel kinetics. (A) Mean open time as a function of membrane voltage in the absence (open circles) and presence of 100 µM NFx (filled circles) in single-channel recordings depicted in Fig. 4. The lines through the data are drawn by hand. (B) Relative areas of the shortest (squares) and the second shortest (circles) closed states in the absence (open symbols) and presence (filled symbols) of 100 µM NFx. (C) Mean shortest closed time as a function of membrane voltage in the absence (open circles) and presence of 100 µM NFx (filled circles). (D) Mean second shortest closed time as a function of membrane voltage in the absence (open circles) and presence of 100 µM NFx (filled circles). (E) Distribution of open times in the absence (open bars) and presence (filled bars) of 100 µM NFx at −100 mV. (F) Distribution of open times in the absence (open bars) and presence (filled bars) of 100 µM NFx at +100 mV. (G) Distribution of closed times in the absence (open bars) and presence (filled bars) of 100 µM NFx at −100 mV. (H) Distribution of closed times in the absence (open bars) and presence (filled bars) of 100 µM NFx at +100mV.

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  Figure 6.. The relative charge of NFx does not contribute to its inhibitory effects. (A) Electrostatic profile of a K+ through the pore of the TREK-2 channel in the presence and absence of NFx. The relevant region where NFx binds just below the filter is expanded below and shows two independent calculations in the presence of NFx compared with the Apo structure. A minor increase in the barrier for K+ permeation is seen in the presence of two charged NFx molecules bound at their sites below the filter. (B) Single-channel recording of TREK-2 channel at −40 mV in an excised patch from HEK293 cells in either the absence or presence of an uncharged NFx derivative (10 µM desamino chloro-fluoxetine) as indicated. Dotted lines in top traces represent the closed-channel level. A similar outcome was obtained in five separate experiments. (C) Histograms of single-channel currents from recordings shown above. For clarity, both amplitude histograms were scaled to the same open-channel level. The chemical structure of desamino chloro-fluoxetine is shown on the left.

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  Figure S6.. NFx affects open state of WT TREK-2 channels expressed in HEK cells. (A) Single-channel recordings of TREK-2 in the excised patch at −10 mV in the absence (top trace) and presence of 10 µM NFx (bottom trace). The dotted line represents zero current level. (B) Dwell-time distributions of channel openings in the absence (black bars) and presence (red bars) of 10 µM NFx. The lines are the best fit of the data to a single exponential function.