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. 2002 Jun 17;21(12):2968-76.
doi: 10.1093/emboj/cdf288.

An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1

Eric Honoré  1 , François Maingret, Michel Lazdunski, Amanda Jane Patel
Affiliations

An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1

Eric Honoré et al. EMBO J. .

Abstract

The 2P domain K(+) channel TREK-1 is widely expres sed in the nervous system. It is opened by a variety of physical and chemical stimuli including membrane stretch, intracellular acidosis and polyunsaturated fatty acids. This activation can be reversed by PKA-mediated phosphorylation. The C-terminal domain of TREK-1 is critical for its polymodal function. We demonstrate that the conversion of a specific glutamate residue (E306) to an alanine in this region locks TREK-1 in the open configuration and abolishes the cAMP/PKA down-modulation. The E306A substitution mimics intracellular acidosis and rescues both lipid- and mechano-sensitivity of a loss-of-function truncated TREK-1 mutant. We conclude that protonation of E306 tunes the TREK-1 mechanical setpoint and thus sets lipid sensitivity.

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Figures

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Fig. 1. Alanine scanning of the proximal C-terminal region of TREK-1. (A) AA strongly potentiates WT TREK-1 current amplitude. (B) Current amplitudes were measured at rest and after stimulation for 2 min with 10 µM AA at a membrane potential of 0 mV in a physiological K+ gradient. The number of cells studied is indicated. The S300, T303, E306 and E309 mutants have a significantly higher basal current compared with TREK-1 WT (indicated by an asterisk). A total of 0.1 µg of DNA was transfected per plate for WT and mutants. (C) AA has a minor effect on the E306A mutant. In (A), (B) and (C), currents were measured with voltage ramps of 600 ms in duration from a holding potential of –80 mV in a physiological K+ gradient.
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Fig. 2. Single-channel properties of TREK-1 WT and E306A mutant. (A) Single-channel activity was recorded at –80 mV in the excised inside-out patch configuration in a symmetrical K+ gradient. In the basal condition, the channel open probability is extremely low (Po = 0.006) and only very few brief openings are detected for TREK-1 WT. (B) The amplitude histogram constructed with a 10 s recording demonstrates that TREK-1 WT channels are essentially closed at rest. (C) After addition of 10 µM AA to the internal medium, robust channel activity is detected for TREK-1 WT (Po = 0.45). (D) The basal channel activity of E306A recorded at –80 mV in a symmetrical K+ gradient is extremely high compared with TREK-1 WT (Po = 0.81). (E) The amplitude histogram constructed with a 10 s recording demonstrates that the open channel probability of E306A is elevated in the non- stimulated condition. (F) Addition of 10 µM AA fails to significantly affect E306A channel activity (Po = 0.84). (G) Single-channel I–V curves of TREK-1 WT (n = 9) and E306A (n = 14). The single-channel amplitude of E306A is slightly smaller than TREK-1 WT at negative and positive potentials. The single-channel conductances estimated in the linear range of the I–V relationships are 130 and 104 pS for TREK-1 WT and E306A, respectively. A total of 0.005 µg of DNA was transfected per plate in these experiments. Currents were filtered at 5 kHz and sampled at 20 kHz. The external solution was free of divalent cation.
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Fig. 3. The E306A mutant is insensitive to membrane stretch, intracellular acidosis and AA. (A) Membrane stretch of –66 mm Hg reversibly opens TREK-1 WT channels in inside-out macro patches. (B) The same membrane stretch fails to affect E306A. (C) Intracellular acidosis from pH 7.2 to pH 5.0 reversibly opens TREK-1 in an inside-out patch. (D) Intracellular acidosis from pH 7.2 to pH 5.0 reversibly reduces E306A current amplitude. (E) Intracellular acidosis to pH 5.0, 10 µM AA and stretch of –66 mm Hg strongly stimulate TREK-1 WT, while failing to enhance E306A current in inside-out patches. These experiments were performed in a physiological K+ gradient at a holding potential of 0 mV. The number of patches studied is indicated and the effect of pH 5.0, AA and stretch on TREK-1 WT is significantly different from E306A (indicated by an asterisk).
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Fig. 4. The side chain at position 306 is critical for TREK-1 gating. (A) Substitution of E306 with hydrophobic, positively and negatively charged residues differentially affects channel opening. For each mutant, the current was recorded in the absence and in the presence of 10 µM AA. The currents were recorded in the whole-cell configuration and the amplitude was measured at 0 mV in a physiological K+ gradient. (B) The E306C, but not the E306A mutant is reversibly blocked by intracellular Cd2+ in the inside-out patch configuration. The free concentration of Cd2+ in the intracellular medium was 100 µM. Voltage ramps of 600 ms in duration were applied from a holding potential of –80 mV in a physiological K+ gradient (inset). The currents were recorded in inside-out macro patches, and the mean current amplitude was measured at 0 mV. The number of patches studied is indicated and statistical difference is indicated with an asterisk.
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Fig. 5. E306A mutation rescues the mechano-sensitivity of a loss-of-function C-terminal truncated TREK-1 mutant. (A) Excised inside-out patches expressing the Δ100 mutant (the last 100 amino acids in the C-terminal region have been deleted) were stimulated by a membrane stretch of –66 mm Hg at both intracellular pH 7.2 (left panel) and pH 5.0 (right panel). (B) Effects of an intracellular acidification from pH 7.2 to pH 5.0 on mean current amplitude recorded at both atmospheric pressure (white) and at –66 mm Hg (black) in inside-out patches expressing the Δ100 TREK-1 mutant (n = 14). The difference between stretch-activated channel activity at pH 7.2 and pH 5.0 is significant (indicated by an asterisk). (C) Relationships between Δ100 TREK-1 (n = 4) mean current amplitude and membrane negative pressure (mm Hg). The experimental data were fitted with a Boltzmann function. The intracellular pH was lowered from 7.2 (open symbols) to 5.0 (filled symbols). The pressure value required to elicit 50% of channel activity is estimated as –48 mm Hg at pH 5.0. (D) Effect of an intracellular acidification from pH 7.2 (left panel) to pH 5.0 (right panel) on the Δ100/E306A mutant at both atmospheric pressure and at –66 mm Hg (stretch). (E) Effects of an intracellular acidification from pH 7.2 to pH 5.0 on mean current amplitude recorded at both atmospheric pressure (white) and at –66 mm Hg (black) in inside-out patches expressing the Δ100/E306A TREK-1 mutant (n = 9). (F) Relationships between TREK-1 WT (n = 21), Δ100 TREK-1 (n = 5) and Δ100 TREK-1/E306A (n = 10) mean current amplitude and membrane negative pressure (mm Hg). The experimental data were fitted with a Boltzmann function. The pressure values required to elicit 50% of channel activity were –38 and –49 mm Hg for TREK-1 WT and Δ100 TREK-1/E306A, respectively. In these experiments, the holding potential was 0 mV and currents were recorded in a physiological K+ gradient.
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Fig. 6. Opening of TREK-1 by the protonophore FCCP critically depends on the presence of E306. (A) In a mock-transfected COS cell, 5 µM FCCP does not activate a K+ current, but occasionally activates a small non-selective current reversing at a reversal potential of about –15 mV. (B) FCCP (5 µM) strongly activates TREK-1. In (A) and (B), the holding potential was –80 mV and cells were stimulated by voltage steps from –160 to 120 mV with a 40 mV increment. (C) FCCP (3 µM) opens TREK-1, whereas blockers of the mitochondrial electron transport chain [cyanide (3 and 10 mM) and rotenone (10 µM)] do not significantly affect TREK-1. (D) FCCP opens TREK-1 dose dependently and the EC50 is 2.1 µM. (E) Effects of 5 µM FCCP on TREK-1, Δ100, E306A and Δ100/E306A mutants. The effect of FCCP on mutant channels is not significant compared with mock-transfected cells. (F) Effects of 10 µM AA on TREK-1, Δ100, E306A and Δ100/E306A mutants. The difference between the Δ100 and the Δ100/E306A mutants is significant (indicated by an asterisk). Currents were recorded in the whole-cell configuration at a membrane potential of 0 mV in a physiological K+ gradient.
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Fig. 7. E306A mutation impairs cAMP-mediated down-regulation of TREK-1. (A) Extracellular addition of the membrane-permeant CPT-cAMP derivative (0.5 mM) produces a rapid and strong inhibition of whole-cell TREK-1 currents elicited in the presence of a HCO3–-rich solution (superfused for 2 min). The holding potential was –80 mV and the cell was depolarized to 100 mV. (B) Basal as well as stimulated (AA, HCO3–) TREK-1 channel activities are down-regulated by CPT-cAMP. However, E306A and S333A are resistant to this treatment. The conservative mutation E306D is as sensitive to cAMP as TREK-1 WT. Currents were recorded in the whole-cell configuration at a membrane potential of 0 mV in a physiological K+ gradient. (C) E306A mutant is resistant to CPT-cAMP (0.5 mM). Voltage ramps of 600 ms in duration were applied from a holding potential of –80 mV in a physiological K+ gradient.
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Fig. 8. (A) Helical wheel representation of the proximal C-terminal domain of TREK-1 (from G293 to F310). The residues sensitive to the alanine substitution, S300, T303, E306 and E309, are indicated in black. (B) Functional model of TREK-1 gating. TREK-1 flickers between two closed states and one open state. When E306 is protonated the channel is mechanically coupled to the bilayer (closed state 2). In this conformation, mild mechanical stimulation or chemical stimulation by polyunsaturated fatty acids such as AA reversibly induces TREK-1 opening. At acidic intracellular pH, TREK-1 opens at atmospheric pressure because of high mechano-coupling to the membrane. The E306A mutation mimics intracellular acidification and opens TREK-1 at atmospheric pressure. In contrast, at alkaline intracellular pH, when E306 is deprotonated, the channel becomes mechanically uncoupled (closed state 1) and is more resistant to membrane stretch as well as lipid stimulation. Similarly, phosphorylation of the PKA site S333 or partial truncation of the C-terminal domain (Δ100) locks TREK-1 in the mechanically uncoupled closed state 1.
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References

    1. Belardetti F. and Siegelbaum,S.A. (1988) Up- and down-modulation of single K+ channel function by distinct second messengers. Trends Neurosci., 11, 232–238. - PubMed
    1. Bockenhauer D., Zilberberg,N. and Goldstein,S.A. (2001) KCNK2: reversible conversion of a hippocampal potassium leak into a voltage-dependent channel. Nature Neurosci., 4, 486–491. - PubMed
    1. Buckler K.J. and Vaughan-Jones,R.D. (1998) Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells. J. Physiol., 513, 819–833. - PMC - PubMed
    1. Duprat F., Lesage,F., Fink,M., Reyes,R., Heurteaux,C. and Lazdunski,M. (1997) TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J., 16, 5464–5471. - PMC - PubMed
    1. Duprat F., Lesage,F., Patel,A.J., Fink,M., Romey,G. and Lazdunski,M. (2000) The neuroprotective agent riluzole activates the two P domain K+ channels TREK-1 and TRAAK. Mol. Pharmacol., 57, 906–912. - PubMed

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