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Figure 1. The down state can be conductive. (A, left) Structure of TREK-2 in the down state showing NFx (as colored spheres) bound within the fenestrations (Dong et al., 2015). K+ ions within the filter are shown in magenta. (right) Cartoon model illustrating how activation by membrane stretch reduces inhibition by NFx (red pentagon). In the unbound down state (middle), the filter gate is closed. The NFx-binding site is only found in the down state; thus, channel activation by membrane stretch (up state, blue) reduces availability of the NFx-binding site. This binary two-state model of gating assumes the down state is always closed and the up state is always open. (B) Relative position of the filter gate mutations (G167I and W306S) that directly activate TREK-2 and the Y315A mutation in TM4, which affects movement between the down and up states. (C) All three mutations produce similar increases in basal whole-cell currents recorded at 100 mV (n ≥ 20; left) and similar reductions in fold activation by membrane stretch (right); currents were recorded in inside-out patches at 0 mV before and after application of negative pressure. Error bars shown are mean ± SEM (n = 5). (D) Example traces showing inhibition by 30 µM NFx inhibition for WT and mutant channels. The voltage protocol involves a step to 0 mV from a holding potential of −80 mV, followed by a ramp from −100 to 60 mV. (E) Dose–response curves showing NFx inhibition is unaffected by the filter gate mutations (G167I and W306S) but reduced by the Y315A mutation. WT IC50 4.9 ± 0.5 µM, n = 16 compared with G167I (IC50 7.0 ± 0.6 µM, n = 10), W306S (IC50 5.0 ± 0.4 µM, n = 10), and Y315A (IC50 > 30 µM, n = 8). Error bars shown are mean ± SEM (n ≥ 6). (F) Revised gating model showing that channels activated by the filter gate mutations are in the down state, whereas activation by the Y315A mutation affects movement between states to reduce NFx inhibition. Image published in: McClenaghan C et al. (2016) © 2016 McClenaghan et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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