PLD2 dependent mechanical activation of TREK-1 channels.

(a-b) Representative traces from pulled patches of human TREK-1 over expressed in HEK293T cells with phospholipase D2 (PLD2, green traces) (a) or catalytically dead PLD2 (xPLD2, red) (b) with pressure clamp (0-60 mmHg at +30 mV). (c) The data, minus HEK293 background (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 truncated TREK-1 (TREK1 trunc) lacking the PLD2 binding site (p=0.002, n=15-23) The inset compares mock transfected HEK293T cells with TREK-1 trunc and full length (FL) TREK1 + xPLD2, indicative of direct TREK-1 activation. Astricts are relative to TREK1 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. TREK-1 control (ctrl) is a C-terminal truncation (C321) of TREK-1 found to have very little current. (e) Cartoon showing PLD2 dependent TREK-1 opening. TREK-1 senses force causing the channel to favor an open conformation (black arrows). When PLD is active it makes phosphatidic acid (PA) which maintains the open state. If PA is not present the channel is not maintained in the open state.

Shear moves TREK-1 nanoscopic distances in HEK293 cells.

(a) A shear fixing protocol is shown. Cell growing in a shear chamber are fixed while shear is applied. Fixed samples are then labeled with fluorescent antibodies or CTxB and imaged for nanoscopic movement (<250 nm) by two-color super resolution imaging and pair correlation (Pair corr.). (b) Combined PALM-dSTORM imaging of TREK1_eGFP and Alexa 647 cholera toxin B (CTxB) with and without shear in HEK293T cells. The middle panel, outlined in grey, is the zoomed portion of the cell surface outlined in the top panel. The bottom panel is a zoom of the cell surface from a separate cell treated with shear. Locations of TREK-1/GM1 proximity are outlined with a white circle. (c) Pair correlation of TREK1 with GM1 before and after shear (3 dynes/cm2; green) determined by combined eGFP-dSTORM imaging when mouse phospholipase D2 (mPLD2) is over expressed (non-permeabilized). The significance in the Pair corr. change is shown across the range of radii 50-70 nm (along the curve) and at a single 50 nm radius (inset). (d) combined eGFP-dSTORM imaging of TREK-1_eGFP with Alexa 647 labeled PIP2 in the presence of over expressed mPLD2 (permeabilized). Significance is shown for radii 70-85 nm along the curve and at a single 225 nm radius (inset). (e-f) Combined PALM-dSTORM of TREK-1 in the presence of catalytically dead PLD2 (xPLD2). Shear (3 dynes/cm2) of TREK-1 is shown as a red curve with xPLD2 present. The experiments are as described in panels c-d. In (e) a significant shift in TREK-1/GM1 pair correlation is shown for 50-70 nm (along the curve) and at a 50 nm radius (inset). In (f) pair correlation did not appear to shift significantly. Significance was determined by a student’s t test or for multiple point a nested student’s t test; *p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001. (g) Cartoon showing TREK-1 movement from GM1 lipids (top) toward PIP2 lipids (bottom) in response to mechanical shear (red arrow).

Shear mobilizes PLD2 within ordered GM1 lipids.

(a) two-color dSTORM images of fixed C2C12 cells with and without (3 dynes/cm2) shear. Cells were labeled with fluorescent CTxB (ganglioside GM1) or antibodies (anti-PIP2 or PLD2 as indicated) and sheared with the temperature held constant at 37 ℃. Scale bar = 1 µm. (b) Cross pair correlation (unitless) of PLD2 with GM1 or PIP2 lipids at a given radius. Error bars are shown at each point radii. A bar graph (inset) is shown at the shortest calculated radii of 5nm. Prior to shear (grey line) PLD2 is clustered with GM1; after shear there is almost no clustering. (c) The opposite was true for phosphatidylinositol 4,5 bisphosphate (PIP2). Prior to shear, PLD2 is not associated significantly with PIP2 clusters, after shear PLD2 clustering dramatically increases. (d) Cluster analysis of the GM1 (d) and PIP2 (e) lipids shown in (a). Shear failed to eliminate GM1 or PIP2 domains after 3 dynes/cm2 shear force. However, their diameter decreases suggesting force deforms but does not eliminate lipid clusters. (e) Cluster analysis of GM1 domains in neuroblastoma 2a (N2a). (f) Fluorescent cholesterol assay. N2a cells grown in a 48 well plates were sheared with 3 dynes/cm2 orbital fluid shear, fixed with shear (10 min), and compared to control cells with no shear using a fluorescent cholesterol assay. After shear, a second set of control cells were allowed to recover with no shear and fixative for 30s (recovery), otherwise the cells were treated identical to experimental cells. (g) A live PLD activity assay shows fluid shear (3 dynes/cm2) increases substrate hydrolysis in cultured N2a cells. (** is p<0.01, **** is p<0.001, **** is p<0.0001, n>800 clusters from 5-6 cells). (h) Depiction of shear thinning activating PLD2. (left) The palmitates of PLD2 (green lines) are shown bound to the palmitate site in ordered GM1 lipids. (right) After shear cholesterol is reduced and the GM1 lipids are deformed. The deformed surface no longer binds palmitates with the same high affinity, and the palmitates is free to move— a process known as shear thinning.

Astrocyte cholesterol regulates TREK-1 through GM1 clustering.

(a) Uptake of cholesterol into cultured cells using the cholesterol transport protein apolipoprotein E (apoE) Panel (b) shows cholesterol/lipid uptake into C2C12 cells with 4 µg/ml (∼110 nM apoE, purple line. Cholesterol dramatically increases TREK-1 pair correlation of TREK-1 with GM1 labeled lipids. Without cholesterol (grey line) very little TREK-1 was clustered with GM1 lipids. Scale bars = 1 µm. (d) Current densities from whole cell patch clamp recordings are shown with and without cholesterol loading with 4 µg/ml apoE in HEK293 cells over expressing human TREK-1. Increasing cholesterol inhibited the channel ∼3 fold (unpaired student’s t test; *p<0.05) (e) Proposed model for cluster associated TREK-1 activation and inhibition. In high cholesterol, TREK-1 clusters with PLD2 and ordered (thick) GM1 lipids inhibiting the channel. In low cholesterol, TREK-1 is partially clustered with PIP2 generating basal TREK-1 activity (see also Fig. 1C). After shear GM1 clusters (dark grey) are deformed further increasing PLD2 and TREK-1 clustering with PIP2 lipids (blue).

Osmotic stretch activates PLD2 in N2a cells.

(a) Stretch by osmotic swell (70 mOsmo buffer) increased PLD2 catalytic activity in live N2a cells compared to isotonic control cells (310 mOsm) (n=5, p<0.001). (b-c) dSTORM imaging showing PLD2 trafficking from ganglioside GM1 to PIP2 clusters in response to 70 mOsm stretch in N2a cells. Cells were treated, fixed, and labeled with anti-PIP2 or PLD2 antibody or cholera toxin B (CTxB, GM1). Prior to stretch PLD2 clustered with GM1 lipids and very little with PIP2 (grey lines). After stretch PLD2 clustered robustly with PIP2 but very little with GM1 lipids suggesting PLD2 trafficked from liquid ordered domain (Lo), to the liquid disordered domain (Ld). (d) Proposed model for TREK-1 stretch activation in a biological membrane by a cluster associated protein activation mechanism (CAPA) and direct force from lipid (FFL). TREK-1 in the open conformation (green ions), in complex with PLD2, is shown after stretch (large red arrows) trafficking (curved black arrow) to PIP2 clusters (blue shaded bars) in the thin liquid disordered (Ld) region of the membrane (light grey). A known gating helix (grey cylinder) is shown in the up (open channel) position with a PLD2 binding site immediately following the helix (green tube). The opening is a response to three factors that combine to raise the gating helix to the up position. 1) FFL (small red arrows) in TREK-1 favors an open (up) helix conformation. 2) The tip is brought into proximity of PLD2 in the open position and 3) PA (red sphere) is produced and maintains the up positioned by binding to charged residues (blue tube) pulling the helix toward the membrane.

PA modulates mechanosensitivity in D. melanogaster.

(a) Shear (3 dynes/cm2) robustly activates PLD2 in a live PLD assay with cultures neuronal insect cells. (b) Cholera toxin robustly labels GM1 lipids (GM1, green), throughout the brain of D. melanogaster. (left). Zoomed section (right) shows that most of the labeling is found on the membrane. There are notable variations in the amount of CTxB labeling with some cells expressing GM1 over the entire membrane (black arrows) while some cells only have labeling in small puncta (white arrow). (c) Measurements of D. melanogaster mechanosensation in vivo. Animals with or without the pldnull gene were stimulated by increasing amounts of mechanical vibration (see supplemental Fig. S4). Flies with lacking PLD2 had a decreased threshold (i.e., more sensitivity to mechanical stimulation) compared to genetically matched controls (w1118) (p=0.039, n= 28-29), consistent with the prediction that PA decreases excitability of nerves. (d)The same result was observed in a PLDRNAi line which results in PLD knockdown only in the neurons of Drosophila (p=0.002, n=28-29). (e) Flies were subjected to increasing voltages of electrical shock in a two-choice assay. PLD-KD flies showed an increased sensitivity to shock when compared with wild-type flies. PLDRNAi flies had a higher aversion to shock at 10V (p=0.0213, n=21) and 20V (p=0.0492, n=27-30), but not at 30V (p=0.672, N=12) (f) (g) Proposed role of PLD2 in regulating mechanical thresholds. PA is a signaling lipid in the membrane that activates hyperpolarizing channels and transporters. When PA is low the membrane is less polarized, and cells are more sensitive to mechanical activation. The downstream targets are unknown (shown with a ‘?’). Flies do not have a TREK-1 homolog.

The role of lipids and lipid order in mechano-transduction.

The plasma membrane is composed of lipids which can cluster into separate and distinct domains with unique properties such as thickness and charge (a). These domains contain proteins which localize to them through post-translational acylation (b). Domain localization exposes proteins to micro-environments which can regulate their activity. For example, our previous findings showed that PLD is localized to GM1-labeled domains due to its palmitoylation sites (c, top). This sequesters PLD from high concentrations of substrate (phosphatidylcholine) and activating lipid (PIP2). Upon chemical or mechanical disruption of the domain, the environment changes, instead favoring PLD translocation and activation (b, bottom) by substrate presentation. This process is a form of cellular transduction. (d) Transduction is known to occur directly between a mechanical to chemical signal using raft-localized enzymes, but it is unknown whether electrical transduction also occurs directly or can be facilitated through a secondary mechanism.

Expression and staining of TREK-1 in cell culture.

(a) Confocal imaging of HEK293T cells over expressing an eGFP tagged human TREK-1. Cells were fixed and stained with anti-trek antibody and a secondary cy3b antibody and A647 conjugated cholesterol toxin B (CTxB). Without transfection no GFP is visible. A negative control (no primary antibody) has no fluorescence. (b) Over expressed eGFP-TREK-1 in HEK293 cells (identical to those used in Fig. 1, can be seen in the plasma membrane of each construct used for electrophysiology. (c-d) over expressing catalytically dead PLD2 (xPLD), appears to slightly increase the amount of TREK-1 expression. Truncating the c-terminus (TREK trunc) has no effect on expression at the plasma membrane.

Electrophysiology details and methods.

(a) Diagram of the C-terminal end of TREK-1 showing the truncation site (red) used and the predicted PLD binding site for PLD2. The last transmembrane helix (M4) is shown as a grey cylinder, and the anionic lipid binding site is highlighted in blue. (b) Individual cell traces for TREK-1+PLD2 (green), TREK-1+xPLD2 (red), and TREK-1-truncated (grey) current densities (pA/µm2). (c) The pressure required to open TREK-1 is decreased when PLD is overexpressed (p<0.05, n=15-20). The observation is coming from very little current (red traces in panel a). (d) Representative cell showing TREK-1 pressure currents (0-60 in 10 mmHg increments). Bottom left is activation step, bottom right is deactivation. Both activation and inhibition appear to be sub 5 ms processes, which is at the limit of detection for our instrument setup. (e) Membrane inactivation. After stretch, the membrane relaxes and palmitates from PLD2 regel with the GM1 lipids and TREK is pulled into GM1 clusters through its interaction with PLD2 (see Fig. 3e for evidence of a complex in GM1 clusters). Absent PA and due to increased hydrophobic thickness of the membrane, the gate is in the down (closed position, marked with an X). (f) Direct inactivation of TREK-1 through an intermediate. Release of stretch could in theory force the channel into a closed conformation by a large amount of direct pressure on the channel (large red arrows). In a thin membrane this would cause the helix to move up to 8 Å away from the membrane and potentially disrupt the PLD2/TREK-1 interaction allowing PLD2 to translocate back to GM1 lipids. This would be an intermediate as TREK-1 would likely re-associate in the thick lipids.

Comparisons of labeling type and permeabilization.

(a) dSTORM with cy3b labeled TREK-1 antibody after shear in the presence of over expressed mouse PLD2 (mPLD2) with full length human TREK-1. HEK293T cells were fixed, permeabilized, and stained with a Cy3b conjugated anti-TREK-1 antibody. Pair correlation (Pair corr.) of TREK-1 with A647 conjugated cholera toxin B (CTxB) was determined by two-color dSTORM and decreased with 3 dynes/cm2 orbital fluid shear. (b) The same experiments as (a) is shown using pair correlation from the eGFP C-terminally expressed with TREK-1 instead of cy3b labeled anti-TREK-1 antibody. The eGFP in in dSTORM buffers produced a robust dSTORM signal. The data also provides a direct comparison of permeabilized cells vs. non-permeabilized seen Fig. 2b. Pair Corr. of TREK-1 with GM1 decreased dramatically decreased in three conditions. (c) A comparison of cy3b-STORM with eGFP-STORM seen in (Fig. 2e) in the presence of xPLD2. (d-e) A comparison of non-permeabilized (a) vs. permeabilized (b) HEK293T cells over expressing full length human TREK-1 and endogenous PLD2 (enPLD2), i.e., no over expression of PLD. (f) TREK-1/PIP2 Pair correlation increased slightly after shear. The insets show, in a-d, the variability at a single radius. Statistics for single points were calculated with a student’s t test. Statistical comparisons at multiple radii were made with a nested student’s t test. (g) Schematic of brain slices mounted for dSTORM. Mouse brains fixed by whole body perfusion were sliced, labeled with cy3b-anti-TREK1 antibody and A647 CTxB and placed on a cover slip with fiberglass filter paper on top to hold the tissue in place during imaging. dSTORM buffer was added to the filter paper. The fluorescent background (640 nm) was undetectable at saturating intensities of light. (h) example images of brain slices from control and astrocyte specific SREBP2 null mice. TREK-1 was expressed in most areas of the brain, but not all (left panel). Pair correlation was determined from regions with both TREK-1 and CTxB labeling. The slice is coronal section near the hippocampus, but the exact region of interest is unknown.

Observing cellular changes in response to mechanical stimulation.

(a) Representative images showing the effect of shear on the apparent size of GM1 (maroon and green) in C2C12 and N2a cells respectively. PIP2 clusters in muscle C2C12 cells are shown in blue. When shear force is applied the apparent size decreases. Scale bars = 1 µm. (b) cluster analysis of PIP2 show a small but significant decrease. (c) TREK-1 and PLD2 are complexed before and after shear in C2C12 cells. TREK-1 and PLD2 cross pair correlations are shown before (grey line) and after (red line) 3 dynes/cm2 shear. Their association remains almost identical in both states. (d) TREK-1 staining with phosphatidylinositol 4,5 bisphosphate (PIP2) antibody in C2C12 cells. Before shear (grey line) TREK-1 associated significantly with PIP2, after shear the association further increased, suggesting some TREK-1 complex moved to PIP2 clusters in C2C12 cells. (e) Shear pair correlation of TREK-1 with GM1 clusters (CTxB) measured by dSTORM in C2C12 cells prior to cholesterol loading showing in Fig. 4b. In low cholesterol endogenous TREK-1 clusters very little with GM1 lipids. (f) Shear thinning model for PLD2. Cholesterol is shown packing with saturated lipids and saturated palmitate via Van der Waals interaction (within 5 nm). Unsaturated lipids contain a double bond that changes to packing surface of a lipids. In a perfectly ordered state palmitoylated proteins are ordered with the GM1 lipids and cholesterol. In the disordered region palmitates are fluid within the membrane. After shear the GM1 lipids remain ordered but deformed. The palmitates no longer pack efficiently with the GM1 lipids, decreasing their affinity for the ordered domain and allowing the palmitates to move in the membrane. (g) Fluorescence recovery after photobleaching (FRAP) imaging to examine potential labeling artifacts57,61,62 of pentavalent CTxB that could remain after fixation. The duration of bleaching is shown in grey. Blue line shows that PFA and glutaraldehyde fixation is sufficient to restrict the large-scale movement of lipids in fixed cells when compared to live cells (red). (h) For both PIP2 and GM1 labeling, shear did not decrease the overall counts with dSTORM significantly in C2C12 cells (p > 0.05, Student’s t-test).

Pan-neuronal knockdown of pld in Drosophila results in a change in sensitivity to shock.

(a) PLD in N2a cells show an increase in activity when subjected to mechanical force. (b) Mechanical stimulation was applied in a series of six increasing vibrations (top). Vibration motor was attached to the back of the chamber with the flies and flies were monitored using a web camera for stimulation-induced arousal (bottom).

Proposed Lipid mixing model of TREK-1 mechanosensation.

In domain-mediated mechanosensation, in an unstimulated state, domains are intact, isolating the PLD2/TREK-1 complex from their respective activating lipids (PC/PIP2, top). After stimulation, the raft is disrupted, causing the PLD2/TREK-1 complex to translocate (right) and eventually bind to PC and PIP2. Upon binding, PLD2 is activated (bottom) transducing the mechanical signal to a chemical signal which then binds to and activates TREK-1 in chemical-to-electrical transduction. Upon stimulation removal, the domain reforms (left) causing the complex to be re-sequestered, causing a return to steady-state conditions (top).