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Graphical Abstract
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Figure 1. Distinct basal and mechanically activated open states in TRAAKWT
(A) Macroscopic currents recorded across an inside-out patch pulled from a TRAAK-expressing cell in response to a voltage step protocol (Vhold = 0, Vtest = −80 to 80, ΔV = 10 mV, 20 mV intervals displayed) with a pressure step applied at each voltage (purple bar).
(B) Current-voltage relationship from (A).
(C) Open probability calculated from single-channel records (0.04 ± 0.02, 0.47 ± 0.07, and 0.96 ± 0.01 for low PO, mid PO, and high PO, respectively [mean ± SEM, n = 3, 4, and 4 patches]). All single-channel data in the paper were recorded at Vhold = 0 mV in a 10-fold gradient of [K+] and are presented in physiological convention.
(D–F) 1 s portion from representative (D) low PO, (E) mid PO, and (F) high PO recordings, respectively.
(G–I) All event (left) and open-only or square root all event (right) current histograms from representative (G) low PO, (H) mid PO, and (I) high PO recordings, respectively.
(J) Unitary currents of O1 and O2 states (0.81 ± 0.03 pA and 1.80 ± 0.16 pA, 0.99 ± 0.03 pA and 2.14 ± 0.09 pA, and 1.07 ± 0.07 pA and 2.41 ± 0.13 pA for low PO, mid PO, and high PO recordings, respectively).
(K) Open dwell time of O1 and O2 states (0.80 ± 0.15 ms, 0.91 ± 0.05 ms and 3.52 ± 0.51 ms, and 9.60 ± 1.46 ms for low PO, mid PO, and high PO recordings, respectively).
(L) Closed dwell times of C1 and C2 states (28.70 ± 13.50 and 0.77 ± 0.03 ms, 3.59 ± 0.67 and 0.58 ± 0.05 ms, and 0.35 ± 0.01 ms for low PO, mid PO, and high PO recordings, respectively). For (J)–(L), data are mean ± SEM, n = 3, 4, and 4 patches; n.d., not determined; n.s., not significant; ∗∗∗p = 0.0006, ∗∗∗∗p < 0.0001 (one-way ANOVA with Tukey correction).
(M) Unitary current-open dwell time relationships for low PO, mid PO, and high PO open events and an overlay of low PO and high PO relationships at expanded timescale. Bubble size is proportional to percentage of open events.
(N) Four-state TRAAK gating model.
See also Figures S1–S5 and Table S1.
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Figure 2. The FHEIG mutation TRAAKA198E promotes a long-duration, high-conductance TM4 up open state
(A) Crystal structure of TRAAKA198E. Side view from the membrane plane with one protomer gray and the second protomer colored according to the key below. A198E is shown as a yellow sphere, and K+ ions are colored purple.
(B) Macroscopic currents from a TRAAKA198E-containing patch in response to a voltage step protocol (Vhold = 0, Vtest = −80 to 80, ΔV = 10 mV, 20 mV intervals displayed) with a pressure step applied at each voltage (dark blue bar).
(C) Current-voltage relationship from (B).
(D and E) Open probability calculated from all (D) TRAAKA198E and (E) TRAAKA198E with pressure (+P) records (left, PO = 0.90 ± 0.04 and PO = 0.97 ± 0.003 [mean ± SEM, n = 4 patches]) and 1 s portion from representative recordings.
(F and G) All event (left) and square root all event (right) current histograms from representative (F) TRAAKA198E and (G) TRAAKA198E+P recordings.
(H) Unitary currents of TRAAKWT HPO, TRAAKA198E, and TRAAKA198E+P O1 and O2 states (1.07 ± 0.07 and 2.41 ± 0.13 pA, 1.44 ± 0.14 and 2.57 ± 0.10 pA, and 1.35 ± 0.06 and 2.72 ± 0.09 pA, respectively).
(I) Open dwell times of TRAAKWT HPO, TRAAKA198E, and TRAAKA198E+P O2 states (9.60 ± 1.46 ms, 4.63 ± 1.03 ms, 12.46 ± 1.02 ms, respectively).
(J) Closed dwell times of TRAAKWT HPO, TRAAKA198E, and TRAAKA198E+P C2 (0.35 ± 0.01 ms, 0.42 ± 0.02 ms, and 0.39 ± 0.01 ms, respectively). For (H)–(J), data are mean ± SEM, n = 4 patches; n.d., not determined; n.s., not significant (one-way ANOVA with Dunnett correction).
(K) View of the membrane-facing lateral opening in a TRAAKWT TM4-down structure (PDB: 4WFF). A cavity-bound lipid acyl chain blocks conduction.
(L) TRAAKA198E in the same view as (K). A TM4-up conformation seals the membrane opening.
(M) Ions in a TRAAKA198E-Tl+ structure. Anomalous density (gray) around Tl+ ions (green) displayed at 2.5 σ (extracellular/selectivity filter/cavity ions).
(N) Overlay of the TM2-TM3-TM4 interaction from TRAAKA198E and TRAAKWT structures. A198E sterically and electrostatically promotes a TM4-up open state.
See also Figures S1–S7 and Tables S1 and S2.
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Figure 3. The FHEIG mutation TRAAKA270P promotes a long-duration, high-conductance TM4-up open state
(A) Crystal structure of TRAAKA270P. Side view from the membrane plane with one protomer gray and the second protomer colored according to the key below. A270P is shown as a yellow sphere, and K+ ions are colored purple.
(B) Macroscopic currents from a TRAAKA270P-containing patch in response to a voltage step protocol (Vhold = 0, Vtest = −80 to 80, ΔV = 10 mV, 20 mV intervals displayed) with a pressure step applied at each voltage (dark green bar).
(C) Current-voltage relationship from (B).
(D and E) Open probability calculated from all (D) TRAAKA270P and (E) TRAAKA270P with pressure (+P) records (left, PO = 0.94 ± 0.01 and PO = 0.96 ± 0.01 [mean ± SEM, n = 6 and 5 patches]) and 1 s portion from representative recordings.
(F and G) All event (left) and square root all event (right) current histograms from representative (F) TRAAKA270P and (G) TRAAKA270P+P recordings.
(H) Unitary currents of TRAAKWT HPO, TRAAKA270P, and TRAAKA270P+P O1 and O2 states (1.07 ± 0.07 and 2.41 ± 0.13 pA, 1.34 ± 0.15 and 2.65 ± 0.16 pA, and 1.21 ± 0.09 and 2.57 ± 0.12 pA, respectively).
(I) Open dwell times of TRAAKWT HPO, TRAAKA270P, and TRAAKA270P+P O2 states (9.60 ± 1.46 ms, 8.56 ± 1.69 ms, and 14.47 ± 3.30 ms, respectively).
(J) Closed dwell times of TRAAKWT HPO, TRAAKA270P, and TRAAKA270P+P C2 (0.35 ± 0.01 ms, 0.43 ± 0.01 ms, and 0.39 ± 0.01 ms, respectively). For (H)–(J), data are mean ± SEM, n = 4, 6, and 5 patches; n.d., not determined; n.s., not significant (one-way ANOVA with Dunnett correction).
(K) View of the membrane-facing cytoplasmic half of TM4 in TRAAKA270P. A TM4-up conformation seals the membrane opening.
(L) Ions in the TRAAKA270P-K+ structure. Polder omit Fo-Fc density (gray) around K+ ions (purple) displayed at 5 and 5.5 σ for extracellular and selectivity filter and cavity ions, respectively.
(M) Overlay of TM4 from TRAAKA270P and TRAAKWT structures. A270P kinks TM4 to promote a TM4-up open state.
See also Figures S1–S7 and Tables S1 and S2.
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Figure 4. The gain-of-function mutation TRAAKG158D promotes a short-duration, low-conductance TM4-down open state
(A) Crystal structure of TRAAKG158D. Side view from the membrane plane with one protomer gray and the second protomer colored according to the key below. G158D is shown as a yellow sphere, and K+ ions are colored purple.
(B) Macroscopic currents from a TRAAKG158D-containing patch in response to a voltage step protocol (Vhold = 0, Vtest = −80 to 80, ΔV = 10 mV, 20 mV intervals displayed) with a pressure step applied at each voltage (dark orange bar).
(C) Current-voltage relationship from (B).
(D) Maximum fold activation by pressure of macroscopic TRAAKWT, TRAAKG158D, TRAAKA198E, and TRAAKA270P currents with expanded scale in inset (119.8 ± 45.38, 1.91 ± 0.14, 1.12 ± 0.02, and 1.16 ± 0.02, respectively, mean ± SEM, n = 9, 11, 7, and 8, ∗∗p < 0.0021, ∗∗∗∗p < 0.0001 [one-way ANOVA with Dunnett correction]).
(E and F) Open probability calculated from all (E) TRAAKG158D and (F) TRAAKG158D with pressure (+P) records (left, PO = 0.71 ± 0.04 and PO = 0.89 ± 0.02 [mean ± SEM, n = 3 patches]) and 1 s portion from representative recordings.
(G and H) All event (left) and square root all event (right) current histograms from representative (G) TRAAKG158D and (F) TRAAKG158D+P recordings.
(I) Unitary currents of TRAAKWT LPO, MPO, HPO, TRAAKG158D, and TRAAKG158D+P O1 and O2 states (0.81 ± 0.03 pA and 1.80 ± 0.16 pA, 0.99 ± 0.03 pA and 2.14 ± 0.09 pA, 1.07 ± 0.07 pA and 2.41 ± 0.13 pA, 0.99 ± 0.01, and 1.06 ± 0.02 and 2.02 ± 0.08 pA, respectively).
(J) Open dwell times of TRAAKWT LPO, MPO, HPO, TRAAKG158D, and TRAAKG158D+P O1 and O2 states (0.80 ± 0.15 ms, 0.91 ± 0.05 ms and 3.52 ± 0.51 ms, 9.60 ± 1.46 ms, 0.88 ± 0.07 and 3.58 ± 0.46 ms, and 6.63 ± 0.83 ms, respectively).
(K) Closed dwell times of TRAAKWT LPO, MPO, HPO, TRAAKG158D, and TRAAKG158D+P C1 and C2 states (28.70 ± 13.50 and 0.77 ± 0.03 ms, 3.59 ± 0.67 and 0.58 ± 0.05 ms, 0.35 ± 0.01 ms, 0.42 ± 0.01 ms, and 0.76 ± 0.03 ms, respectively). For (I)–(K), data are mean ± SEM, n = 3, 4, 4, 3 and 3 patches; n.d., not determined; n.s., not significant, ∗∗∗∗p < 0.0001 (one-way ANOVA with Tukey correction).
(L) Unitary current-open dwell time relationships for TRAAKG158D, and TRAAKG158D+P open events and an overlay at expanded timescale. Bubble size is proportional to percentage of open events.
(M) View of the membrane-facing cytoplasmic half of TM4 in TRAAKG158D.
(N) Ions in the TRAAKG158D-K+ structure. Polder omit Fo-Fc density (gray) around K+ ions (purple) displayed at 6.5 and 6 σ for selectivity filter and extracellular and cavity ions, respectively.
(O) The conduction path in TRAAKG158D colored by hydrophobicity. G158D increases cavity electronegativity to promote a TM4-down open state.
See also Figures S1–S7 and Tables S1 and S2.
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Figure 5. An integrated model for TRAAK gating with distinct basal and mechanically gated open states
Known structures are mapped to the linear four-state model for TRAAK gating. Basal activity corresponds to a TM4-down, low-conductance, short-duration open state O1. Mechanically gated activity corresponds to a TM4-up, high-conductance, long-duration open state O2. Long-duration closures correspond to a TM4-down lipid-blocked state C1. The unknown structure of the short-duration closed state C2 is drawn without ions, and the position of TM4 is indicated with dashed lines.
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