Mammalian TRP ion channels are insensitive to membrane stretch

1Transient receptor potential (TRP) ion channels constitute a superfamily of non-selective cationic channels permeable to Na+, Ca2+ and Mg2+, comprising 33 members found in mammals, of which 27 are found in humans. TRP channels are divided into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin) and TRPA (ankyrin). These channels play a significant role in sensory physiology, with most of them contributing to cellular Ca2+ signaling and homoeostasis. All TRP channels are polymodally regulated and involved in various sensations in humans, including taste, temperature, pain, pressure and vision. Multiple studies have demonstrated TRP channel involvement in mechanosensory transduction in mammals, including most notably TRPA1, TRPV4, TRPV2, PKD2, PKD2L1, TRPC3 and TRPC6.

2Although TRP channels have been reported to be involved in mechanosensory processes, in most cases it remains controversial whether they are the primary transducers of mechanical force. Mechanosensitivity of TRP channels is only understood well for no mechanoreceptor potential C (NOMPC; also known as TRPN) channels and the first such evidence came from a Caenorhabditis elegans study. NOMPC, whose homologs are not found in mammals, has also been shown to be activated by mechanical force pulling on the 29 ankyrin repeats that act as tethers associated with microtubules. In Drosophila, NOMPC is involved in many mechanosensitive processes, including gentle touch in larvae. In contrast, other TRP channels, such as TRPV4, clearly respond to mechanical force but their mechanisms for doing so are subject of debate. Although the deletion of ankyrin repeats in TRPV4 does not eliminate the mechanosensitivity of the channel, it does reduce its sensitivity to hypotonicity. TRPV4 also contributes to mechanotransduction in chondrocytes and bone, and – in this context – the channel does not seem to respond to membrane stretch. Thus, although it is clear that TRP channels are components of mechanosensory systems, whether they are primary mechanosensors that respond to membrane stretch remains contentious.

3Here, we systematically explore the stretch sensitivity for several representatives of all six mammalian TRP channel superfamily members that are heterologously expressed in HEK293T cells and most of which have been linked to mechanosensory transduction. In total, we investigate eleven TRP channels, including TRPC3, TRPC5, TRPC6, TRPM4, TRPM8, TRPV1, TRPV3, TRPV4, TRPA1, PKD2L1 and TRPML1. In addition, to examine the role the heterologous system itself has in channel activity, we tested TRPC6 sensitivity in response to membrane stretch in Chinese hamster ovary (CHO), mouse Neuro2a (N2a) and human HeLa cell lines, and purified channels reconstituted into liposomes and recording their activity by using the patch clamp technique. Our results indicate that none of the TRP channels we tested, including TRPV4, can be activated directly by membrane stretch.

4To investigate whether mouse TRPC6 responds to mechanical stimuli in vivo, we examined the behavior of a transgenic C. elegans-expressed mouse TRPC6 on the background of a strain (i.e. osm-9) that is insensitive to mechanical stimuli. This osm-9 strain lacks the gene encoding the C. elegans ortholog of the mammalian TRPV4 channel. We show that mouse TRPC6 is able to restore the nose-touch response in osm-9 worms but requires diacylglycerol (DAG); suggesting that TRPC6 acts downstream of a mechanosensory pathway. We comprehensively illustrate the lack of stretch activation in the examined TRP channels and discuss our results in relation to the literature reporting TRP channels involvement in mechanosensory transduction processes.

5Given the important role that TRP channels play in mechanosensory processes, we sought to determine whether these ion channels are sensitive to membrane stretch. We did this through the patch clamp technique, which is a electrophysiological technique is used to record activity of ion channels characterized by opening frequency of the single channels as well as ion currents flowing through the channels in response to a voltage and/or negative pressure (suction) applied to a patch clamp pipette. Initially, we recorded the activity of TRPC family members TRPC3, TRPC5 and TRPC6 in response to negative pressure pulses that had been applied to cell-attached patches for 300 ms (Fig. 1B–D). Channels were expressed in HEK293T cells and channel identity was confirmed by their ion conductance and, in the case of TRPC6, pharmacological properties (Figs S1 and S2, Table 1). We found that negative pipette pressure did not increase the probability of ‘open state’ (hereafter referred to as open probability) for any of the TRPC ion channels examined (Fig. 1E). As a positive control, we also expressed the well-known mechanosensitive channel Piezo1 in HEK293T cells and found that stretch could elicit large inactivating currents that are characteristic of this ion channel (Fig. 1F). In addition, we also evaluated stretch activation of TRPC6 in three widely used heterologous expression systems, i.e. CHO, HeLa and N2a cells (Fig. 1G), to examine whether the cellular background plays a role in channel sensitivity in response to membrane stretch. In all three cell types, however, membrane stretch failed to increase the open probability of TRPC6 channel activity (Fig. 1I). Our data, thus, indicate that this member of the TRPC ion channel subfamily is insensitive to membrane stretch. Importantly, in these experiments we used wild-type HEK293T and N2a cells that express endogenous Piezo1 channels. Patches in which endogenous Piezo1 activity was detected were excluded from analysis.

6We applied a similar approach to test the stretch sensitivity of other TRP family members, i.e. TRPA1, TRPM4, TRPM8, TRPV1, TRPV3, TRPV4, PKD2L1 (TRPP2; formerly TRPP3) and TRPML1 (surface-expressing mutant) in HEK293T cells. We recorded the spontaneous resting activity of all tested channels that did not exhibit any change in response to membrane stretch (Fig. 2B–I). The channel identities were confirmed by their characteristic conductance (Figs S3, S4, S5 and Table 1). In each case, the channel open probability was not influenced by applied pipette pressure (Fig. 2J). Taken together, these data provide no evidence of stretch sensitivity in any of the TRP channels expressed in heterologous systems and examined in this study.

7A defining feature of inherently mechanically activated ion channels is that they can be activated by stretch in artificial lipid bilayers. Given the possibility that some components present in the heterologous systems inhibit stretch-activation of the TRP channel currents, we purified and reconstituted the TRPC6 channel protein into liposomes (Fig. 3) to examine the channel response to bilayer tension induced by applied pipette pressure. The purified TRPC6 protein appeared as a single monodisperse peak on a gel filtration column and as a single band on denaturing SDS-PAGE gels, indicating purity of the channel (Fig. 3A,B). For these experiments we used, in addition to WT, an N-terminal truncation (Δ94) of TRPC6, whose channel properties have been reported to be very similar to the WT channel. Previously, the Δ94 TRPC6 truncation was used for the structural determination of the TRPC6 cytoplasmic domain.

8In addition to WT TRPC6 channels, we also observed spontaneous openings of Δ94 TRPC6 channels expressed in liposomes, with conductance values similar to those of TRPC6 channel expressed in cells (Fig. 4). Remarkably, the WT TRPC6 channel was significantly more active in liposomes than in cells (Fig. 4B,C,F,G). TRPC6 has been reported to be an outward rectifier – i.e. passing current more easily out of the cell – in whole cell mode ( preprint). When recording single-channel activities in cell-attached mode, we found that positive holding potentials slightly decreased single-channel conductance (Fig. 4B) and increased the open probability of the channel (Fig. 4D), which explains the outward-rectifying currents observed in the whole-cell recordings. We also found that, like in cells, open probability and conductance of the recorded channels in liposomes were modulated by voltage (Fig. 4 B–D,F–H). Conductivity of full-length TRPC6 reconstituted into liposomes was comparable to that of its truncated version used in this study (Fig. S6). Collectively, our data demonstrate that TRPC6 assembled into a pore-forming oligomer in liposomes, where it exhibits biophysical properties comparable to those exhibited by channels expressed in cells. The observation that TRPC6 is inwardly rectifying in liposomes, strongly suggests that the channel incorporates unidirectionally into the artificial bilayer – with the cytoplasmic domain facing the external bath solution (Fig. 3C), similar to what has been reported for several other reconstituted ion channels.

9We further examined the mechanosensitivity of TRPC6 reconstituted into liposomes by applying mechanical stretch to excised patches. We found that spontaneously active TRPC6 was insensitive to the application of negative pressure (Fig. 5B) and its open probability was unaffected by stretching the liposome bilayer (Fig. 5D). In contrast, stretching the well-characterized mechanosensitive channel MscL reconstituted into liposomes, activated the channel and increased its open probability (Fig. 5C,E). Application of stretch to empty liposome patches in control experiments did not elicit any currents (Fig. 5A). These results demonstrate that, activation of TRPC6 is unaffected by the changes in bilayer tension, comparable to the results obtained with the channels expressed in heterologous cell systems.

10TRPC6 has been reported numerous times to be involved in mechanosensory processes. However, whether TPRC6 is a primary mechanotransducer still remains to be determined. We implemented C. elegans as an in vivo model to determine the role of TRPC6 in mechanotransduction. In this animal model, it is well-established that mammalian TRPV4 can restore touch sensitivity in osm-9 worms (a mutant strain insensitive to nose-touch stimuli; Fig. 6A) when expressed in ASH sensory neurons, i.e. the neurons that are required in C. elegans for a wide range of avoidance behaviors – and by stroking the nose of the worm with an eyelash. OSM-9 is the C. elegans orthologue of mammalian TRPV4; noteworthy, OSM-9 acts downstream of the DEG-1 channel in the ASH-mediated mechanical response.We expressed Mus musculus (m)TRPC6 (no TRPC6 ortholog is encoded in the C. elegans genome) in ASH sensory neurons to determine whether TRPC6 can recover the touch response of osm-9 worms (Fig. 6A). We found that, upon mechanical stimulation, mTRPC6 could restore the withdrawal response of osm-9 worms, similar to that in the wild-type (N2) worms (Fig. 6A), establishing that the channel is functional in ASH neurons (Fig. 6A). However, when reducing the DAG content by feeding phospholipase C (PLC) inhibitor (u73122) to the worm (Fig. S7A), mTRPC6 lacks the ability to recover touch sensitivity. It is noteworthy that feeding worms an inactive PLC inhibitor analog (u73343) did not affect TRPC6-mediated response (Fig. S7C). PLC enzymes are known to obtain DAG from the inner membrane monolayer, which in turn activates TRPC6 via GPCR pathway. Therefore, our results suggest that mTRPC6 is acting downstream of a mechanosensory pathway that generates DAG but not as a direct mechanosensor. To test whether the DAG content also modulates the chemically induced response of TRPC6, we challenged mTRPC6 transgenic worms with the TRPC6 specific agonist GSK1702934A (Fig. 6B). We found that GSK1702934A elicited dose-dependent robust withdrawal responses (Fig. S7B) in mTRPC6-expressing but not in wild-type or TRPV1 transgenic worms (Fig. 6B). Unlike the mechanical response, inhibition of PLC did not affect the withdrawal behavior mediated by chemical activation of mTRPC6 (Fig. 6B), since the agonist directly activates the channel.

11In this study we investigated the mechanosensitivity of eleven TRP channels from all six families of the TRP ion channel superfamily heterologously expressed in HEK293T cells, examining their activity in response to stretching of the cell membrane in patch clamp experiments. In addition to TRPC3, TRPC5 and TRPC6, we also examined TRPM4, TRPM8, TRPV1, TRPV3, TRPV4, TRPA1, PKD2L1 and TRPML1. We focused further on TRPC6 by examining the stretch sensitivity of this channel when expressed in three other cell lines, i.e. CHO, HeLa and N2a cells. We also purified the TRPC6 channel protein and reconstituted it into liposomes to examine its stretch sensitivity in isolation. The key observation from these experiments is that none of the examined TRP channels exhibited increased activation in response to a membrane stretch of 300 ms, indicating that none of these channels are directly activated by membrane tension. As a control, we used the well-characterized mechanosensitive Piezo1 and MscL channels, which under the same experimental conditions exhibited robust stretch-activated ion currents in cell and liposome membrane patches, respectively.

12Overall, our results contrast with the results reported in many studies describing TRP channel activation by membrane stretching, which has been reported numerous times to activate TRP channels: TRPV4, TRPA1, PKD1/PKD2 (TRPP2), TRPC1, TRPM4, TRPC5 and TRPC6. Notably, inherent mechanosensitivity – which would suggest TRP channels to be primary mechanosensors – was never provided for any of these channels. In agreement with our findings, some studies do not support the inherent mechanosensitivity of TRPC6, TRPV4 and TRPM4. Given that cells are highly heterogeneous systems, with diverse protein and lipid composition, the cellular environment can be expected to play an essential role in the function of mechanosensitive, i.e. stretch-activated, ion channels. Therefore, considering the heterogeneity of expression systems as a possible cause for the discrepancies between different reports, we tested the mechanosensitivity of TRPC6 channels in several other types of cell as well as in proteoliposome patches. Despite the absence of the cellular environment in proteoliposome patches we were unable to record TRPC6 activation in response to membrane stretch. This result is consistent with our results concerning the other TRP channels whose membrane-stretch sensitivity we investigated in HEK293T cells. Consequently, given our results and the evidence provided by many studies reporting the involvement of different types of TRP ion channels in cellular mechanotransduction processes, we conclude that most mammalian TRP channels are not stretch-sensitive primary mechanosensors but are more likely to be secondary membrane receptors involved in downstream mechanosensory signaling cascades. Indeed, we showed that mouse TRPC6 can act downstream of a mechanosensory pathway in C. elegans; however, it requires DAG to recover the mechanical response.

13We cannot, however, completely exclude the possibility that some of these TRP channels, nevertheless, function as primary mechanoreceptors if – like NOMPC channels – they are tethered directly to the cytoskeleton or extracellular matrix. As secondary downstream mechanoreceptors, these channels can contribute to either an increase of intracellular Ca2+ (e.g. TRPC3, TRPC6, TRPV1, TRPV4, PKD2L1) or the depolarization of the membrane potential through inward Na+ and/or Ca2+ currents (e.g. TRPM4, TRPA1). In some cases, where TRP channels are Ca2+-dependent, they may be directly downstream of Ca2+ permeable mechanosensitive channels such as Piezo1. In other instances, transduction of the mechanical force to the channel may occur through other mechanosensing proteins. For example, GPCRs can be activated by mechanical force and produce a host of secondary messengers (e.g. cAMP, cGMP, Ca2+, DAG) that activate TRP channels through ligand–channel interactions. One scenario for Gq-coupled receptors is the cleavage of PIP2 to produce DAG through activation of PLC. In this case, there are three possible scenarios for how this may stimulate TRP channel opening. First, DAG might act as a ligand opening the channel. Second, PIP2 might have an inhibitory influence, thus its removal will allow the channel to open. Third, the shape change instigated by conversion of PIP2 to DAG might generate local membrane curvature in the vicinity of a TRP channel causing it to open, as demonstrated for TRP and TRPL channels in Drosophila melanogaster. Such a scenario has been proposed for TRPC6 channels, given that local membrane curvature can generate enough stress in the membrane bilayer to activate mechanosensitive channels. Indeed, as has been previously shown, we were also able to activate TRPC6 channels in response to the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) (Fig. S2). This indicates that ion channel mechanosensitivity exists in different forms, with local membrane curvature being one of them. Although membrane stretch and local membrane curvature are mechanical stimuli that activate mechanosensitive channels, such as MscL, MscS, TREK-1, TRAAK and Piezo1, this does not seem to be the case for other ion channels, such as TRPC6, which may be able to differentiate between these two kinds of mechanical stimulus. Further to this point, it is important to note that even homologues of the prototypical mechanosensitive channel MscL exhibit a different sensitivity to membrane stretch and curvature, despite their high degree of sequence similarity. Thus, it is eminently feasible that channel structure influences stretch and curvature sensitivity of an ion channel.

14Further to our results that demonstrate insensitivity to membrane stretch of mammalian TRP channels, we also demonstrated, for the first time, a successful functional reconstitution of TRPC6 ion channels in a model lipid system without any other protein components present. TRPC6 ion channels were spontaneously active and exhibited high open probability in liposomes. They were ∼40 times more active in liposomes compared to within HEK293T cell patches. Spontaneous ion channel activities upon reconstitution into artificial lipid bilayers have also previously been reported for Piezo1 channels. Furthermore, the lack of TRPC6 responsiveness in liposomal membranes further confirms that not all proteins reconstituted in lipid bilayers are gated by membrane stretch.

15In conclusion, we have demonstrated that mammalian members of different subfamilies of TRP channels are insensitive to membrane stretch. This does not preclude their involvement in cellular mechanotransduction processes but does suggest, in many cases, that they do not represent the primary mechanotransducers. If any one of them is, in fact, a primary mechanotransducer, then its mechanosensory abilities are likely to be dependent on auxiliary transmembrane proteins or tethers that link them to structural scaffold proteins. However, we showed that activation of transgenically expressed mouse TRPC6 in C. elegans is DAG dependent and were able to activate TRPC6 in vitro with OAG. Both reagents have been proposed to generate local bilayer curvature in the vicinity of the channel. From these results we conclude that the mechanosensitivity of TRP channels can be different compared to that of other well-known mechanosensitive ion channels, including MscL and Piezo1. This suggests that controversies regarding the mechanosensitivity of the TRP ion channels originate from the fact that different mechanical stimuli, such as membrane stretch, shear force or local membrane curvature, do not affect all mechanosensitive channels in the same way. The structure of some channels, including the TRP ion channels, might have, in fact, evolved to discriminate between different mechanical stimuli based on their interactions with other membrane and cellular components.

16HEK293T, N2a, HeLa and CHO cells were used for heterologous expression of TRP channels. Cells were grown in Dulbecco's modified Eagle medium (DMEM) with 10% FBS, then transferred on 2 ml Petri dishes for transfection. Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) kit was used according to manufacturer instructions: 1–3 μg of plasmid (Table S1), 5 μl of Lipofectamine 3000, and 5 μl of P3000 were mixed in 250 μl Opi-MEM (Gibco) for 5 min and then added to cells for 24 h incubation. Some plasmids were mixed with 0.5 μg GFP (pIRES2-EGFP) to enable selection of transfected cells. The surface-expressing TRPML1 mutant TRPML1-L15L/AA-L577L/AA (4A) was used for patch clamp recordings. All plasmids were verified by Sanger Sequencing.

17Protocol for mTRPC6 purification was used from a previous publication. Two versions of mTRPC6 were purified: 94 aa truncated (Δ94 mTRPC6) and the full-length (WT) protein. Both versions were 8His-maltose binding protein (MBP) tagged on the N- terminus. DNA constructs were cloned into the pFastbac1 plasmid. Recombinant baculovirus was generated (Bac-to-Bac expression system; Invitrogen) and transfected into Sf9 cell culture (800 ml) for 72 h. Cell pellets were resuspended and disrupted with a homogenizer (Avestin) in a solution containing 36.5 mM sucrose, 50 mM Tris, 4 mM TCEP (pH 8), and protease inhibitors: 1 mM PMSF, 1 mg/ml pepstatin, 3 mg/ml aprotinin, and 3 mg/ml leupeptin. Unbroken cells were removed by centrifugation at 8000 g for 15 min. Supernatants were further ultracentrifuged (Beckman) at 100,000 g for 30 min at 4°C. Pellets were solubilized in 26 mM DDM (Anatrace), 150 mM NaCl, 10% Glycerol, 50 mM HEPES, 2 mM TCEP (pH 7.4), and protease inhibitors by rotating for 2 h. Then the mixtures were ultracentrifuged at 150,000 g for 45 min, and the supernatants were incubated with amylose resin (New England Biolabs) for 3 h. The resins were loaded onto the column and washed with 10 volumes of 0.5 mM DDM, 150 mM NaCl, 10% glycerol, 50 mM HEPES, and 2 mM TCEP (pH 7.4). The proteins were eluted with same solution supplemented with 20 mM maltose. The MBP tags were cleaved with ProTEV Plus protease (Promega) at 4°C overnight. Proteins were further purified by Superose 6 10/300 GL column (GE Healthcare) running in a solution containing 150 mM NaCl, 20 mM HEPES, and 2 mM TCEP (pH 7.4). The peak fractions corresponding to the tetrameric mTRPC6 channel were collected for proteoliposome reconstitution.

18Liposome reconstitution of the TRPC6 and MscL proteins was carried out using modified method as previously published. L-α-Phosphatidylcholine from soybean (Sigma-Aldrich, P5638) was stored in chloroform that was evaporated using nitrogen before use. The lipids were resuspended in 200 mM KCl and 5 mM HEPES (pH 7.2) at a final concentration of 10 mg/ml. The mixture was vortexed for 5 min and sonicated for 15–30 min until the liposome suspension became translucent. Purified mTRPC6 was mixed with the lipids (1:100 protein-lipid w:w) and incubated for 1 h at room temperature. Bio-Beads SM2 resin (Bio-Rad, 1523920) was used to remove detergent by incubating (15 mg/ml) with the protein lipid mixture for 3 h at room temperature. The supernatant was ultracentrifuged at 150,000 g (Beckman) for 30 min. The pellet was resuspended in 30 µl of solution containing 200 mM KCl and 5 mM HEPES (pH 7.2), then spotted onto the clean glass slide, and dried in the desiccator overnight at room temperature. The next day, samples were hydrated with the same solution and further incubated overnight at 4°C. Proteoliposomes (5 µl) were added to the recording chamber and incubated for 15 min before patching. For TRPC6: bath solution contained: 140 mM NaCl, 10 mM KCl, 0.2 mM CaCl2, and 5 mM HEPES (pH 7.4). Patch pipettes were filled with the same solution. After GΩ seal formation the patch was always excised. Wild-type E. coli MscL ion channel was purified, as reported previously, using immobilized metal affinity chromatography and reconstituted into the liposomes similarly to TRPC6. MscL was recorded in symmetrical bath and pipette solution containing 200 mM KCl, 40 mM MgCl2, and 5 mM HEPES (pH 7.2).

19Channel activity was directly recorded in cells in Petri dishes within 48 h after transfection. Glass recording pipettes (Drummond, 100 μl), were pulled with a vertical puller (Narishige PP-83). A cell-attached or inside-out patch mode was used to record single-channel activity. Resistance of the patch pipettes was in the range of 2–5 MΩ. GΩ seals with the cell membrane were aided by applying ∼10 mmHg negative pressure. Seal formation was monitored by pipette current. A high-speed pressure clamp (ALA Scientific Instruments), connected to the patch pipette allowed rapid pressure application. Single-channel events were recorded with a patch-clamp amplifier Axopatch 200B (Molecular Devices) at 10 kHz sampling rate and filtered at 1–2 kHz with a Digidata® 1440 Digitizer (Molecular Devices) using pCLAMP v10.3 software (Molecular Devices). The voltage values shown in the figures are holding potentials. All patch-clamp recordings were conducted at room temperature (∼22°C). Cells were recorded in the bath solution: 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 3 mM HEPES (pH 7.2). The pipette solution contained 90 mM CsCl, 50 mM CsF, 5 mM HEPES, and 4 mM EGTA (pH 7.2 adjusted with NaOH). For TRPML1 recordings bath solution also contained 1 mM MgCl2; pipette solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM HEPES (pH 7.4). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) (Avanti) and SKF96365 (Cayman Chemical) were dissolved in DMSO and were added to the external solution with final concentration 30 μM and 10 μM, respectively.

20Worm strains were cultured as previously described. The wild-type (N2) C. elegans strain was obtained from the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Transgenic worms were obtained using the MosSCI method. The GN132 strain was a gift from Dr Miriam B. Goodman (Stanford University). A complete list of strains is presented in Table S2. The chemical response test was performed at 21°C by placing a drop containing the TRPC6 agonist GSK1702934A (50 μM; Tocris Bioscience) or control buffer (M13 buffer: 30 mM Tris–HCl pH 7.0, 100 mM NaCl, 10 mM KCl, with 1% ethanol) in front of moving young adult hermaphrodites as described previously. The nose-touch response test was performed by stroking the worm with an eyelash as previously described. Drop or touch trials that elicited reversals of motion were scored as withdrawal responses. The PLC inhibitor u73122 or its inactive analog u73343 (Tocris Bioscience) dissolved in DMSO (10 mM) were added to the nematode growth medium (NGM) to reach a 10 µM concentration.

21Control- and u73122-treated worms (500 worms per condition) were rinsed three times with M9 buffer containing 22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, and 1 mM MgSO4. Worms were frozen in liquid nitrogen to further extract diacylglycerols (DAGs) at the Lipidomics Core Facility at Wayne State University.

22Data were analysed using the Clampfit 10.3 software (Molecular Devices) and GraphPad Prism 7. Mean single-channel amplitudes for each voltage were acquired by fitting Gaussian curves to the current-amplitude histograms. The conductance values for single channels were obtained by calculating the slope of the current voltage plots. The relative open probability (NPo) was obtained using the single-channel search function in Clampfit. For stretch experiments NPo values were calculated before and upon mechanical stretch. For NPo/voltage plots, open probabilities were calculated for 5 s periods at each voltage. Statistical analyses were carried out using paired t-tests when comparing two groups. Data were reported as mean±s.e.m.;*P<0.05 was considered statistically significant. Statistical analyses of Worm withdrawal were carried out using the Mann–Whitney test.