The pore structure and gating mechanism of K2P channels

Abstract

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.

Figure 1

Quaternary ammonium (QA) ions are high-affinity inhibitors of K2P channels. (A) TRESK channel currents expressed in Xenopus oocytes measured at –80 mV in inside-out patches exposed to 5 μM of the indicated QA ions. (B) Application of 100 μM of TEA and TButA and 10 μM TPenA and THexA on TREK-1. (C) Representative current trace for determining the high-affinity block by TPenA in TREK-1. (D–F) Summary of IC₅₀ values for current inhibition measured for TRESK (n⩾5 experiments for each blocker), TREK-1 (n⩾4 experiments for each blocker) and TASK-3 (n⩾3 experiments for each blocker) channels determined for different chain length QA ions (note that for TREK-1, the IC₅₀ for TEA was estimated due to insufficient block at very high concentrations).

Figure 2

QA inhibition of K2P channels most closely resembles open-channel block in Kv channels. (A) Current–voltage relationship (ramp from −80 to +80 mV) in absence and presence of TPenA for TRESK and Kir1.1 channels at indicated concentrations. (B) Voltage dependence of inhibition measured at 0.3 μM, 2.5 mM and 5 μM TPenA for TRESK (n=20), Kir1.1 (n=3) and ShakerΔ6–46 (inactivation removed) (n=11), respectively. (C) IC₅₀ of TPenA inhibition obtained at +40 mV for the indicated channels with high (120 mM) and low (4 mM) extracellular K⁺ from at least four recordings of each channel and [K⁺] concentration. (D) Comparison of IC₅₀ values obtained for different chain length QA ions in TRESK, Kir1.1 and ShakerΔ6–46 channels (at least n=4).

Figure 3

Identification of residues in the TREK-1 pore that affect TPenA block. (A) IC₅₀ values of TPenA inhibition for TREK-1 WT (n=11) channels and indicated mutations (n⩾4). The orange line represents the deviation (s.e.m.) of the IC₅₀ observed for WT channels. Green bars represent mutations thought to contribute directly to the QA binding site and in yellow those mutations which are not part of the binding site. (B) Current trace from the TREK T157C-L189C double mutation that results in a strongly decreased TPenA affinity. (C) Dose–response relationship from experiments such as in (B) fitted to a standard Hill equation for TREK WT, T157C and L189C mutations and the double mutant which dramatically reduces TPenA affinity; IC₅₀ values of 13±1, 123±11, 120±18 and 8149±428 μM for WT (n=10), T157C (n=5), L189C (n=6) and T157C-L189C (n=4), respectively.

Figure 4

The QA ion binding site in TREK-1. TPenA is shown docked into the KvAP-based homology model of TREK-1. Two different side-view images are shown each with one pore-forming unit removed for clarity. The side chains of residues which affect TPenA inhibition are highlighted. Those predicted to directly interact with TPenA are show in green while those which do not appear to contribute directly to the binding site are shown in orange. The right hand panel shows a snapshot of the movie (Supplementary data), which shows a side view of how TPenA (yellow) docks well into the predicted binding site. Residues that affect TPenA block are shown in green. Coordinates for this model are provided in Supplementary data.

Figure 5

The slow kinetics of TREK-1 inhibition by THexA. (A) State-dependent inhibition of ShakerΔ6–46 channels with 20 μM THexA indicative of open-channel block. (B) Time course of TREK-1 inhibition of indicated QA compounds (5 μM THexA, 50 μM TPenA and 5 mM TButA) equalized for better comparison (i.e., 5 mM TButA produced only 60% inhibition) as monitored by a fast piezo-driven application system. (C) The blocking and unblocking time course from experiments as shown in (B) was fitted with a monoexponential function and τ values for QA inhibition (τ_on) and recovery from QA inhibition (τ_off) are plotted (n⩾3 for each blocker). The dotted lines represent the time courses for activation/deactivation on pHi gating as illustrated in the inset (n=7).

Figure 6

pH activation of TREK-1 does not involve the bundle crossing. (A) TREK currents activated by pH 5 during fast application and wash out of 1 μM THexA. (B) Closed channels at pH 8 were switched into a solution containing 1 μM THexA at pH 5 (red trace) and back to pH 8 showing fast pH-mediated opening and relatively slow blocking by THexA. (C) 1 μM THexA is continuously present during the switch from pH 8 to pH 5 (red trace) and the current is already pre-blocked to 50%, no slow block is observed. (D) As the Po for TREK-1 is increased by acidification to saturating levels no change in IC₅₀ for TPenA inhibition is observed (n⩾6 for each pH). (E) In contrast, in ShakerΔ6–46 channels the IC₅₀ for TPenA inhibition decreases as relative current increases with voltage activation (n⩾4 for each voltage). (F) Pressure activation by 15 mm Hg in absence (black trace) and presence of 1 μM THexA (red trace). (G) TREK-1-gating model. Large movements of the C-terminal regulatory domain are translated into small movements of TM helices to gate the channel at the filter and do not involve gating at the helix bundle crossing.

Figure 7

The selectivity filter is involved in pH gating of TREK-1. (A) Activation of TREK-1 channels by indicated intracellular pH, note the small blockage of the current by pH 4.0. (B) Similar experiment for the T157C mutant in P1. (C) Summary of the EC₅₀ values for pH activation of mutations in P1/P2 region of TREK-1 compared with WT channels that is indicated by the dashed line (see Supplementary Figure S5).

Figure 8

Effect of permeant ions on TREK-1 activation by pH. (A) pH activation of TREK-1 channels in the presence of Rb⁺ in comparison with K⁺ as permeating ion, measured at 40 mV. (B) Dose–response curves from experiments such as in (A) for different ionic species indicating a large effect of the permeant ion on pH activation in TREK-1 channels (n⩾4 for each ion). (C) pH sensitivity of Kir1.1 in the presence of K⁺ and Rb⁺ as the conducting ion. (D) Dose–response curves from experiments such as in (C) fitted with standard Hill equation resulted in pH_0.5=6.62±0.068 for K⁺ (n=6) and 6.58±0.017 for Rb⁺ (n=8). (E) No effect of external [K⁺] concentration on the EC₅₀ for pH activation in TREK-1 channels (n⩾6 for each [K⁺] concentration). (F) Comparison of pH activation in TREK-1 WT (EC₅₀=5.9±0.1; n=9) versus S164Y mutant channels (EC₅₀=6.0±0.1; n=6) (a mutation related to extracellular pH sensitivity; Cohen et al, 2008) indicating no effect of this mutation on intracellular pH sensitivity.