Figure 1. PLD2-dependent and independent mechanical activation of TREK-1 channels.

(A, B) Representative traces from pulled patches of human TREK-1 overexpressed in HEK293T cells with mouse phospholipase D2 (mPLD2, green traces) (A) or catalytically inactive mouse PLD2 (xPLD2, red) (B) under pressure clamp (0–60 mmHg at +30 mV). (C) The data, after subtracting HEK293T background current (0.04 ± 0.02 pA/µm² n = 5 [inset]), are summarized for –60 mmHg. Compared to endogenous PLD2, the expression of xPLD2 eliminated the majority of detectible TREK-1 pressure current (p<0.007, n = 16–23), as did a functional truncated TREK-1 (TREK trunc) lacking the PLD2 binding site (p=0.002, n = 15–23). The inset compares mock-transfected HEK293T cells with TREK trunc and full-length TREK-1 (TREK FL)+xPLD2, indicative of direct TREK-1 activation. Asterisks indicate significance relative to TREK FL, except where noted by a bar. (D) Whole-cell TREK-1 potassium currents with and without xPLD2. TREK-1 is expressed and functional in the presence of xPLD2. A nonfunctional C-terminal truncation (C321) of TREK-1 (xTREK) is shown with no appreciable current HEK293T cells. (E) Cartoon illustrating PLD2-dependent TREK-1 opening in HEK293T cellular membrane. On the left, membrane stretch (black arrows) mechanically activates PLD2. When PLD2 is active, it makes phosphatidic acid (PA), which evokes the open state of TREK-1. On the right, in the absence of mechanically generated PA, the closed channels remain closed despite the presence of membrane tension. Statistical comparisons were made with an unpaired Student’s t-test.

Figure 1—figure supplement 1. The role of lipids and lipid order in mechanotransduction.

(A) The plasma membrane is composed of lipids that can cluster into separate and distinct domains with unique properties such as thickness and charge. Domains for saturated gangliosides (GM1) are shown separate from phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5 triphosphate (PIP₃). These domains contain proteins that are targeted to the domain through post-translational acylation. (B) The types of acylation are shown along with their targeting location. (C, top) The enzyme phospholipase D2 (PLD2, green) is shown with its acylation that binds to the saturated ordered site in GM1. The site discriminates palmitoylated proteins from prenylated proteins. This is called the anesthetic/palmitate (AP) site because anesthetics also compete for this site (not shown) (Pavel et al., 2020; Petersen et al., 2020). (C, bottom) Upon chemical or mechanical disruption of the domain, the binding site is disrupted releasing PLD2 from the AP site. PLD2 is then free to bind PIP₂ where it has access to its substrate phosphatidylcholine (PC). (D) Cartoon depicting a mechanically evoked current through a chemical intermediate.

Figure 1—figure supplement 2. Electrophysiology details and methods.

(A) Illustration depicting the C-terminal end of TREK-1. The red region indicates the truncation site used, and the predicted PLD binding site for PLD2 is highlighted. The last transmembrane helix (M4) is depicted as a gray cylinder, and the anionic lipid binding site is highlighted in blue. (B) Individual cell traces showing current densities (pA/µm²) for TREK-1 co-expressed with PLD2 (green), TREK-1 co-expressed with xPLD2 (red), and TREK-1 with a C-terminal truncation (TREK trunc, gray). (C) Half maximal TREK-1 pressure current within a non-saturating pressure range of 0–60 mmHg. Overexpression of PLD2 significantly reduces the apparent pressure required to activate TREK-1 (p<0.05, n = 15–20). (D) Representative cell recording displaying TREK-1 pressure currents in response to pressure steps from 0 to 60 mmHg, taken in 10 mmHg increments. The bottom-left panel illustrates the activation step, and the bottom-right panel shows the deactivation. Both activation and deactivation processes appear to occur within sub-5 ms time frames, near the limit of detection for the experimental setup. (E) Membrane inactivation process. After mechanical stretching, the membrane relaxes, allowing the palmitates from PLD2, to re-associate with the GM1 lipids. Consequently, TREK is drawn into GM1 clusters through its interaction with PLD2. In the absence of phosphatidic acid (PA) and due to an increased hydrophobic thickness of the membrane, the channel’s gate assumes the down (closed) position, marked with an ‘X’. (F) Direct inactivation of TREK-1 through an intermediate. Upon reversal of mechanical stretch (relaxation of the membrane), the channel may transition into a closed conformation due to direct pressure exerted on the channel (indicated by the large red arrows). In a thin membrane, this action could displace the gating helix up to 8 Å away from the membrane, disrupting the PLD2/TREK-1 interaction. This putative intermediate state is expected to be transient as TREK-1 would likely re-associate in thicker lipid regions.