The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension

Abstract

Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K(+) channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.

Keywords: Liposomes; Mechanotransduction; Membrane Bilayer; Patch Clamp; Potassium Channels.

FIGURE 1.

Purification of TREK-1 from yeast light membranes using streptavidin-Sepharose affinity resin. A, analysis of the solubilized and the eluted fractions by SDS-PAGE followed by Coomassie Blue staining. Lane a, yeast light membrane fraction; lane b, solubilized light membrane fraction; lane c, resin after binding and washing; lane d, elution after thrombin addition; lane e, elution as lane d but starting from yeast transformed with an empty vector. Except for the molecular mass marker (M) and the control (20 μl in lane e), 10 μl of each fraction was loaded (corresponding to 50 μg of total proteins in lane a, for example). T: thrombin. B, analyses of the same fractions as in A by Western blot with anti-TREK-1 antibodies. Only 1 μl was loaded in each lane (corresponding to 5 μg of total proteins in lane a, for example), except for 2 μl in lane e. C, effect of the N-glycosidase F on purified TREK-1. Lane a, initial sample (equivalent to lane d above); lane b, N-glycosidase-treated sample (see “Experimental Procedures”). 1*, 2*, 3*: species of BAD-TREK-1; 1–5: species of TREK-1 after removal of the BAD tag by thrombin.

FIGURE 2.

Evaluation of the size of purified TREK-1 by size exclusion chromatography. A, the figure depicts the elution profile at 280 nm from size exclusion chromatography of TREK-1 after affinity chromatography. To evaluate the size of purified TREK-1, the gel filtration column was calibrated under the same conditions using the Bio-Rad Gel Filtration Standards, with the following molecular masses and Stokes radii: thyoglobulin (Tg) M_r 670,000, R_S = 8.6 nm; bovine γ-globulin (Gg), M_r 158,000, R_S = 5.1 nm; chicken ovalbumin (Ov), M_r 44,000, R_S = 2.8 nm; equine myoglobin (My) 17,500, R_S = 1.9 nm; and vitamin B12 (Vt) corresponding to the total volume. The column elution profiles corresponding to purified TREK-1 (continuous line) and to protein standards (interrupted line) were monitored at 280 nm. The purified TREK-1 profile has been enlarged 4-fold. B, the fractions of interest (designated by the bar on the profile) were pooled and subsequently both analyzed by SDS-PAGE stained with Coomassie Blue (left panel) and revealed with anti-TREK-1 antibodies (right panel), prior or after concentration with a centrifuge concentrator. Ten microliters of each sample was loaded on respective lanes. Lane M: molecular mass marker; lane a: sample of purified TREK-1; lane b: pooled fractions after size exclusion chromatography; lane c: same sample as lane b after 10-fold concentration using a centrifuge concentrator. 1–5: species of TREK-1.

FIGURE 3.

Electrophysiological characteristics of the TREK-1 protein reconstituted in giant liposomes. A, representative single channel currents recorded at different membrane potentials, in symmetrical media, as defined below. The close channel state is marked by an arrow. B, current-voltage relationship and ionic selectivity. Closed circles, I-V curves obtained in symmetrical (bath and pipette, 150 mm KCl); open circles, I-V curves obtained in asymmetrical media (bath, 150 mm KCl; pipette, 5 mm KCl). In both cases, the pH was adjusted to 7.4 with 10 mm HEPES-KOH with, in addition, 5 mm MgCl₂ and 2 mm CaCl₂ in the pipette. The chord conductance at 50 mV was 80 pS under symmetrical conditions and 160 pS under asymmetrical conditions.

FIGURE 4.

Reversible inhibition of the TREK-1 channel activity by chlorpromazine. Current recording segments obtained at +40 mV in the excised inside-out configuration under control condition, during application of 25 μm chlorpromazine (CPZ) and after washout, as indicated. The recording was performed under symmetrical ionic conditions as defined in Fig. 3.

FIGURE 5.

Effect of pressure, applied to the pipette, on the activity of TREK-1 reconstituted in liposomes. A and B, single-channel data recorded at +40 mV upon an application of a positive pressure to the pipette (A) and upon successive applications of negative and positive pressures (B). The upper and lower traces present single-channel currents and pressure records, respectively. The applied pressure was zero at the onset of each trace. The recording was performed under symmetrical ionic conditions as defined in Fig. 3.

FIGURE 6.

Pressure dependence of the purified TREK-1 channel. A, channel activity of TREK-1 at different applied positive pressures to the pipette, as indicated for each trace. The membrane potential was +40 mV, and the recordings were performed under symmetrical conditions as defined in Fig. 3. B, open probability (NP_o) versus the applied pressure of the channels, the activity of which is shown in A. The data are obtained from 10–12 s of recording at each pressure.