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Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel

Abstract

Activation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain1,2,3,4,5. TRAAK (also known as KCNK4) ion channels are neuronally expressed members of the two-pore domain K+ (K2P) channel family and are mechanosensitive6. They are involved in controlling mechanical and temperature nociception in mice7. Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer—it is a membrane-tension-gated channel8. However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and non-conductive conformations defined by the presence of permeant ions along the conduction pathway. In the non-conductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2–TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity—an expansion in cross-sectional area up to 2.7 nm2 in the conductive state is expected to create a membrane-tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.

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Figure 1: Structures of TRAAK in non-conductive and conductive conformations.
Figure 2: Redox-dependent activation of a TRAAK double cysteine mutant between TM2 and TM4.
Figure 3: Conformational changes in conductive TRAAK structures expand the channel cross-sectional area.
Figure 4: Model for TRAAK gating and physical basis of mechanosensitivity.

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Acknowledgements

We thank staff at APS beamlines 23-IDB/D, especially R. Sanishvili and S. Corcoran and at 24-IDC/E, especially I. Kourinov, D. Neau and K. Rajashankar for assistance at the synchrotron and members of the MacKinnon laboratory for discussions. S.G.B. is a Howard Hughes Medical Institute postdoctoral fellow of the Helen Hay Whitney Foundation and R.M. is an investigator of the Howard Hughes Medical Institute.

Author information

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Contributions

S.G.B designed and performed the experiments. E.B.C. performed hybridoma cell culture. R.M. supervised the project. S.G.B and R.M. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Roderick MacKinnon.

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The authors declare no competing financial interests.

Additional information

The X-ray crystallographic coordinates and structure factors for the non-conductive conformation of TRAAK in K+ (4WFF) and Tl+ (4WFH) and the conductive conformation of TRAAK in K+ (4WFE) and Tl+ (4WFG) are available at the Protein Data Bank.

Extended data figures and tables

Extended Data Figure 1 The central cavity in conductive and non-conductive TRAAK conformations.

a, b, View from the membrane plane of the TRAAK central cavity in the non-conductive (a) and conductive (b) conformations. The exposed surface of hydrophobic amino acids are colored white, arginine and lysine are blue, glutamate and aspartate are red, and polar residues are green. The positions of K+ ions in the filter are outlined and residue T277 in TM4 is indicated with an asterisk. c, Diameter of the ion conduction pathway as a function of distance through the membrane for non-conductive TRAAK (red), conductive TRAAK (blue) and TWIK1 (grey, PDB: 3UKM). The green box indicates the position of the selectivity filter and dashed grey lines are the approximate boundaries of the lipid membrane. The 10 Å diameter constriction formed partially by T277 is indicated with an asterisk. The pore diameter is larger in TRAAK than in TWIK1 and expands below T277 in the conductive conformation.

Extended Data Figure 2 Reconstituted TRAAK activity in different lipids.

a, Current recorded from TRAAK proteoliposome patches as a function of holding voltage (mean ± s.e.m., n = 9 patches each from three separate experiments). Current through TRAAK reconstituted in phosphatidylcholine lipids with branched acyl chains (1,2-diphytanoyl-sn-glycero-3-phosphocholine, DPhPC) was significantly higher than in non-branched acyl chains (egg l-α-phosphatidylcholine, PC) at each voltage measured (5.0-fold higher at 0 mV, P < 0.0001, Student’s t-test). b, c, Representative recording of pressure (lower trace) activation of TRAAK current (upper trace) in PC (b) or DPhPC (c) lipids. d, Quantification of pressure activation of TRAAK in PC and DPhPC (mean fold pressure activation at 0 mV ± s.e.m., n = 9 patches each from three separate experiments, ***P < 0.0001, Student’s t-test).

Extended Data Figure 3 Representative electrophysiological recordings from wild-type TRAAK and TRAAK(I159C, R284C).

In these experiments and those in Fig. 2, inside-out patches from cells expressing wild-type or mutant TRAAK channels were excised and perfused with reducing bath solution (with 10 mM DTT). After stabilization of the patch (TRAAK channels exhibit a gradual run-up of current following excision to an equilibrium value, for example, Extended Data Fig. 4), the perfusion solution was switched to oxidizing bath solution (no DTT). a, b, Representative voltage family from a TRAAK(I159C, R284C) patch during perfusion of reducing (a) and oxidizing (b) solution. The voltage family protocol is illustrated. c, d, Same as a, b, but from a wild-type TRAAK patch. e, f, Current response (upper) to pressure application (lower) at 0 mV from the same TRAAK(I159C, R284C) patch during perfusion of reducing (e) or oxidizing (f) bath solution. g, h, Same as e, f, but from a wild-type TRAAK patch. Scale is shown between each pair of recordings in reducing and oxidizing bath solutions.

Extended Data Figure 4 Basal activity and tension activation of TRAAK.

a, Whole cell current from a TRAAK-expressing cell during a voltage step protocol in a tenfold gradient of [K+] (EK+ = −59 mV, holding voltage = −80 mV, ΔV = 10 mV, indicated steps shown). Red dashed line indicates zero current level. b, Current–voltage relationship from experiment in a. c, Currents (upper traces) recorded from an outside-out patch excised from the same cell as in a, b. The voltage protocol in a was used with an additional pressure step (lower trace) during each voltage step. d, Current–voltage relationship from data in b (mean current 5 min after patch excision before pressure (red) and peak current during pressure step (grey)) and a recording immediately after pulling the patch (red dashes). The excised patch contains < 1% of the whole cell membrane area, but gives 25% of the whole cell current before and similar current during a pressure step. This is explained by very low basal activity of TRAAK with near-zero membrane tension (whole cell) and channel activation by increasing membrane tension over a broad range (intermediate tension in an excised patch to high tension in a pressurized patch).

Extended Data Figure 5 Detailed view of TM2–TM3 rotation in TRAAK.

Stereo view from the cytoplasm of an overlay of non-conductive (red) and conductive TM2–TM3 rotated (blue) conformations. Amino acids that sterically prevent TM2–TM3 rotation when TM4 is down are shown as sticks. TM2–TM3 rotates about hinges at positions G169 and G205. This rotation can only occur if TM4 is up because amino acids L172, F201 and G205 on TM2–TM3 shift (0.75–2.1 Å) to a position that would sterically clash with amino acids Y271 and V275 on TM4 in a down conformation. Translation of Y271 and V275 3.1–4.1 Å in TM4 up conformations creates space for the TM2–TM3 rotation.

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Pairwise root mean square deviation (Å) between conformationally static regions between TRAAK structures
Extended Data Table 3 Summary of TRAAK channel conformations in crystal structures

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Brohawn, S., Campbell, E. & MacKinnon, R. Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. Nature 516, 126–130 (2014). https://doi.org/10.1038/nature14013

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