We sought to reproduce the mechanically evoked currents reported when TACAN is expressed in cells (Beaulieu-Laroche et al., 2020). Using CHO cells, similar to those used in the original study, we did not observe pressure-evoked currents in excised membrane patches (Figure 1A,B). Background channels that were not sensitive to the pressure steps were observed in CHO cells expressing either TACAN or the M2 muscarinic receptor as a control. Similarly, TACAN expressed in a Piezo1 knockout HEK cell line did not elicit pressure-activated channels (Figure 1C,D). Purified TACAN protein reconstituted into giant unilamellar vesicles (GUVs) of soy L-α-phosphatidylcholine (soy-PC) also did not yield pressure-activated channels in membrane patches isolated from the GUVs (Figure 1E). We note that previously we have successfully recorded mechanosensitive TRAAK channels in GUVs using the identical approach (Brohawn et al., 2014).

When TACAN was expressed, purified, and reconstituted into both GUVs and planar lipid bilayers at high protein-to-lipid ratios (≥1:100, m:m), transient currents were observed, as shown in Figure 2. These currents were insensitive to pressure applied to patches isolated from the GUVs (Figure 1E) and heterogeneous in amplitude (Figure 2A–C). These properties do not resemble aspects of currents from known ion channels but might suggest that TACAN renders the membrane transiently leaky when reconstituted at high protein concentrations.

Alongside the functional characterization, we analyzed the structure of TACAN determined at 3.5 Å resolution using single-particle cryo-EM. Details of the structure determination are given in Materials and methods and Table 1 (Figure 3—figure supplements 1 and 2). TACAN is an α-helical TM protein that forms a symmetric dimer (Figure 3A). The orientation of the protein with respect to the cytoplasm is unknown; however, the charge distribution on TACAN (von Heijne, 1986) as well as the possible presence of an enzyme active site exposed to the cytoplasm (discussed below) suggests the orientation shown (Figure 3B). Each protomer consists of six TM helices (S1–S6), which form a barrel surrounding a tunnel open to the cytoplasm (Figure 3C). Non-continuous density was observed inside the tunnel, suggesting the presence of a small, non-protein molecule (Figure 3—figure supplement 3A). The two protomers of the TACAN dimer bury an extensive surface area of 3049 Ų, mediated through the TM domain as well as two long N-terminal helices that form a coiled coil (Figure 3A, Figure 3—figure supplement 3B).

The DALI three-dimensional structure comparison server (Holm and Rosenström, 2010) identified a homologous protein called ELOVL7, a long-chain fatty acid (FA) elongase (Figure 4; Nie et al., 2021). This enzyme catalyzes the first step in the FA elongation cycle by transferring an acetyl group from malonyl-CoA onto long-chain FA-CoA (Naganuma et al., 2011). As shown in Figure 4A, the TM domain in TACAN is indeed similar to ELOVL7. The tunnel in ELOVL7 is lined by catalytically important histidine residues and contains a covalently linked eicosanoyl-CoA molecule (Figure 4B,C). TACAN conserves two of the four histidine residues (Figure 4B,D). To determine the identity of the small molecule implied by the broken density in the tunnel of TACAN (Figure 4E and Figure 3—figure supplement 3A), we determined the structure of TACAN with His196 and His197 mutated to alanine at 2.8 Å resolution (Table 1, Figure 4—figure supplement 1). Our rationale was that if the His residues are catalytically important – by analogy to ELOVL7 – then their mutation might influence the occupancy of a potential cofactor. The map showed clearer density consistent with a coenzyme A molecule (CoASH) (Figure 4F). Native mass spectrometry (nMS) was used to confirm the identity as CoASH (Figure 5). As shown in Figure 5B, the purified TACAN^{H196A H197A} sample contains a mixture of the 83,237 Da, +767 Da, and +1535 Da mass species, corresponding to an apo form, one and two CoASH bound forms, respectively. After incubation with CoASH, some fraction of the apo form shifts to one and two CoASH bound forms. Additionally, the +767 Da and +1535 Da species are replaced by +795 Da and +1591 Da or +811 Da and +1622 Da species after incubation with the two CoASH analogues S-ethyl-CoA or Acetyl-CoA, corresponding to the CoASH analogue bound forms. In the purified TACAN^WT sample, the apo form is dominant and incubation with S-ethyl-CoA shifts it to one and two analogue bound forms (Figure 5A). Together, these data are consistent with our cryo-EM results and indicate that TACAN is co-purified with endogenous coenzyme A.

It is noteworthy that CoASH binds with different conformations in TACAN from ELOVL7 (Figure 5—figure supplement 1A,B; Nie et al., 2021). In addition, no enzymatic activity was observed for TACAN using a free-CoA detection assay (for details, see Materials and methods), which demonstrated robust activity for ELOVL7 (Figure 5—figure supplement 1C), thus TACAN does not appear to catalyze the same reaction as ELOVL7. If TACAN is a coenzyme A-dependent enzyme, its substrate is unknown.

Homo sapiens or Mus musculus full-length TMEM120A (TACAN, residues 1–343) were cloned into pEG BacMam (Goehring et al., 2014). The C-terminus of the TACAN construct contains a PreScission protease cleavage site and an enhanced green fluorescent protein (eGFP) for purification (TACAN-eGFP). Briefly, bacmid carrying TACAN was generated by transforming Escherichia coli DH10Bac cells with the corresponding pEG BacMam construct according to the manufacturer’s instructions (Bac-to-Bac; Invitrogen). Baculoviruses were produced by transfecting Spodoptera frugiperda Sf9 cells with the bacmid using Cellfectin II (Invitrogen). Baculoviruses, after two rounds of amplifications, were used for cell transduction. HEK293S GnTl^- cells (ATCC, CRL-3022) grown in suspension at a density of ~3 × 10⁶ cells/mL were transduced with P3 BacMam virus of TACAN-eGFP, and inoculated at 37°C. 8–12 hr post-transduction, 10  mM sodium butyrate was added to the culture and cells were further inoculated for 40–48 hr at 30°C. Cells were then harvested by centrifugation, frozen in liquid N₂, and stored at –80°C until needed.

Frozen cells (from 1 L cell cultures) were resuspended in 200 mL hypotonic lysis buffer containing 50 mM Tris-HCl pH 8.0, 3 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mg/mL DNase I, and a protease inhibitor cocktail (1  mM PMSF, 0.1 mg/mL trypsin inhibitor, 1 µg/mL pepstatin, 1 µg/mL leupeptin, and 1  mM benzamidine) for 30 min and centrifuged at 37,500 g for 30 min. The pellets were then homogenized in 20  mM Tris-HCl pH 8.0, 300  mM NaCl, 0.1 mg/mL DNase I, a protease inhibitor cocktail followed by addition of 10 mM lauryl maltose neopentyl glycol (LMNG), 2 mM cholesteryl hemisuccinate (CHS) (for cryo-EM samples), or 1% n-decyl-β-D-maltopyranoside (DM), 0.2% CHS (w/v) (for reconstitution and mass spectrometry samples) to solubilize for 2 hr. The suspension was then centrifuged at 37,500 g for 30 min and the supernatant incubated with 5 mL GFP nanobody-coupled CNBr-activated Sepharose resin (GE Healthcare) for 2 hr (Kubala et al., 2010). The resin was subsequently washed with 10 column volumes of wash buffer containing 20 mM HEPES pH 7.4, 250 mM NaCl, and 0.06% digitonin (w/v) (for cryo-EM samples) or 0.25% DM, 0.05% CHS (w/v) (for reconstitution and mass spectrometry samples). The washed resin was incubated overnight with PreScission protease at a protein to protease ratio of 40:1 (w:w) to cleave off GFP and release the protein from the resin. The protein was eluted with wash buffer, concentrated using an Amicon Ultra centrifugal filter (MWCO 100 kDa), and then injected onto a Superdex 200 increase 10/300 GL column (GE Healthcare) equilibrated with the wash buffer. Peak fractions corresponding to the TACAN dimer were pooled. For cryo-EM study, the pooled fractions were concentrated to 6–7 mg/mL using an Amicon Ultra centrifugal filter (MWCO 100 kDa). All the purification steps were carried out at 4°C.

H. sapiens full-length ELOVL7 (residues 1–281) was cloned into the same vector, expressed, and purified with the same protocol as TACAN in DM/CHS. The final protein concentration was ~2 mg/mL.

Dialysis-mediated reconstitution of H. sapiens TACAN and ELOVL7 into liposome was accomplished according to published protocols with minor modifications (Brohawn et al., 2012; Heginbotham et al., 1999; Long et al., 2007; Tao and MacKinnon, 2008; Wang et al., 2014). Briefly, 20 mg of soy L-α-phosphatidylcholine (soy-PC) was dissolved in 1 mL chloroform in a glass vial and dried to a thin film under argon, rehydrated in reconstitution buffer (10 mM HEPES pH 7.4, 450 mM NaCl, and 2 mM DTT) to 20 mg/mL by rotating for 20 min at room temperature, followed by sonication with a bath sonicator until translucent. 1% DM was then added, and the lipid detergent mixture was rotated for 30 min and sonicated again until clear. Purified TACAN (~3 mg/mL) or ELOVL7 (~2 mg/mL) in DM/CHS and DM-solubilized lipids (20 mg/mL) were mixed at protein-to-lipid (w:w) ratios of 1:20, 1:50, and 1:100, incubated for 2 hr, and then dialyzed against 4 L reconstitution buffer for 4 days with daily exchange at 4°C. Biobeads (Bio-Rad) were added to the reconstitution buffer for the last 12 hr. The resulting proteoliposomes were flash frozen in liquid N₂ and stored at −80°C.

The dehydration/rehydration-mediated blister formation technique was used for generation of GUVs as previously reported (Brohawn et al., 2014). In brief, an aliquot of reconstituted H. sapiens TACAN proteoliposome was thawed at room temperature and spotted onto the 14 mm glass coverslip inside a 35 mm glass-bottomed Petri dish (Mattek; P35G-1.5-14C) as 4–6 similar-sized drops. Spotted proteoliposomes were then dried under vacuum at 4°C for 6 hr followed by rehydration with ~20 μL rehydration buffer (10 mM HEPES pH 7.4, 140 mM KCl). The rehydration was done by sitting the 35 mm Petri dish inside a 15 cm Petri dish lined with wet filter paper overnight at 4°C (~16 hr). 3 mL bath solution (10 mM HEPES pH 7.4, 140 mM KCl, 1 mM MgCl₂) was then added to the 35 mm dish before recording. Blisters were visible after ~10 min and were competent to form high-resistance seals for at least 2 hrs.

CHO-K1 cells (ATCC) and piezo-1 knockout HEK-293T cells (established in this lab) were used for electrophysiology experiments because they have low endogenous mechanosensitive currents (Brohawn et al., 2014; del Marmol, 2016).

Cells were cultured in DMEM-F12 (Gibco) (CHO cells) or DMEM (Gibco) (HEK-293T cells) supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were plated in 35 mm plastic dishes and grown to ~50–60% confluency at 37°C. Right before transfection, culture media was replaced by DMEM-F12 or DMEM with 10% FBS and 2 mM L-glutamine. 1 μg of H. sapiens TACAN-eGFP or the M2 muscarinic receptor (Halo-M2R-eGFP) plasmid (previously established in this lab) was transfected into the cells using FugeneHD (Promega) following the manufacturer’s protocol. Cells were transferred to 30°C after transfection and recordings were carried out 16–18 hr post-transfection. Immediately before recording, media were replaced by the bath solution (10 mM HEPES pH 7.4, 140 mM KCl, 1 mM MgCl₂).

Pipettes of borosilicate glass (Sutter Instruments; BF150-86-10) were pulled to ~2–6 MΩ resistance with a micropipette puller (Sutter Instruments; P-97) and polished with a microforge (Narishige; MF-83). Recordings were obtained with an Axopatch 200B amplifier (Molecular Devices) using excised inside-out patch techniques. Recordings were filtered at 1 kHz and digitized at 10 kHz (Digidata 1440A; Molecular Devices). Pressure application through patch pipettes was performed with a high-speed pressure clamp (ALA Scientific) controlled through the Clampex software. Pressure application velocity was set to the maximum rate of 8.3 mmHg/ms. All recordings were performed at room temperature. Pipette and bath solutions were identical unless otherwise stated: 10 mM HEPES pH 7.4, 140 mM KCl, and 1 mM MgCl₂ (~300 Osm/L).

The bilayer experiments were performed as previously described with minor modifications (Ruta et al., 2003; Wang et al., 2014). A piece of polyethylene terephthalate transparency film separated the two chambers of a polyoxymethylene block, filled with symmetrical buffer containing 10 mM HEPES pH 7.4, 150 mM KCl unless otherwise stated. A lipid mixture of DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine, Avanti, Cat# 850356):POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, Avanti, Cat# 840857) (3:1, w:w) dissolved in decane (20 mg/mL) was pre-painted over an ∼100 μm hole on the transparency film. Voltage was controlled with an Axopatch 200B amplifier in whole-cell mode. The analog current signal was low-pass filtered at 1 kHz (Bessel) and digitized at 10 kHz with a Digidata 1550A digitizer (Molecular Devices). Digitized data were recorded on a computer using the software pClamp (Molecular Devices, Sunnyvale, CA). Experiments were performed at room temperature.

All the cell lines except for Piezo1 knockout HEK-293T, which was previously generated in the lab, were purchased from ATCC, and we have confirmed there is no mycoplasma contamination for all of them.

For both the WT and His196Ala, His197Ala mutant of M. musculus TACAN, purified protein at a concentration of 6–7 mg/mL was mixed with 2.9 mM Fluorinated Fos-Choline-8 (FFC8; Anatrace) immediately prior to grid preparation. 3.5 μL of the mixture was applied onto a glow-discharged Quantifoil R1.2/1.3 400 mesh Au grid (Quantifoil), blotted for 4 s at room temperature (RT) with a blotting force of 2–4 and humidity of 100%, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI).

Cryo-EM data were collected on a 300-kV Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K2 Summit (TACAN^WT), or a K3 Summit (TACAN^{H196A H197A}) direct electron detector and a GIF Quantum energy filter set to a slit width of 20  eV. Images were automatically collected using SerialEM in super-resolution mode. After binning over 2 × 2 pixels, the calibrated pixel size was 1.03 Å with a preset defocus range from 0.7 to 2.1 μm (TACAN^WT), or 0.515 Å with a preset defocus range from 0.8 to 2.2 μm (TACAN^{H196A H197A}), respectively. Each image was acquired as either a 10 s movie stack of 50 frames with a dose rate of 7.54 e/Ų/s, resulting in a total dose of about 75.4 e⁻/Ų (TACAN^WT), or a 1.5 s movie stack of 38 frames with a dose rate of 37.7 e/Ų/s, resulting in a total dose of about 56.6 e/Ų (TACAN^{H196A H197A}).

For TACAN^WT, the image processing workflow is illustrated in Figure 3—figure supplement 1D. A total of 2,071 super-resolution movie stacks were collected. Motion-correction, twofold binning to a pixel size of 1.03  Å, and dose weighting were performed using MotionCor2 (Zheng et al., 2017). Contrast transfer function (CTF) parameters were estimated with Gctf (Zhang, 2016). Micrographs with ice contamination were removed manually, resulting in 1,982 micrographs for further processing. A total of 583,766 particles were auto-picked using Relion 3.1 (Scheres, 2020; Scheres, 2012; Zivanov et al., 2018; Zivanov et al., 2020) and windowed into 256  ×  256-pixel images. Reference-free 2D classification was performed to remove contaminants, yielding 383,719 particles. These particles were subjected to ab initio reconstruction in cryoSPARC-2.9.0 (Punjani et al., 2017), specifying four output classes. The best class with 245,031 particles was selected, then subjected to a resolution-based classification workflow similar to a previous study (Kang et al., 2020). In brief, 40 iterations of global search 3D classification (K = 1) in Relion 3.1 with an angular sampling step of 7.5° was performed to determine the initial alignment parameters using the initial model generated from cryoSPARC. For each of the last five iterations of the global search, a K  =  6 multi-reference local angular search 3D classification was performed with an angular sampling step of 3.75° and a search range of 30°. The multi-reference models were generated using reconstruction at the last iteration from global search 3D classification low-pass filtered to 8, 15, 25, 35, 45, and 55  Å, respectively. The classes that showed obvious secondary structure features were selected and combined. Duplicated particles were removed, yielding 130,491 particles in total. These particles were subsequently subjected to non-uniform refinement with C2 symmetry in cryoSPARC, which resulted in a map with a resolution of 4.5 Å. Iterative cycles of non-uniform refinement in cryoSPARC with C2 symmetry and Bayesian polishing in Relion 3.1 with new training parameters were performed until no further improvement, resulting in a 3.7 Å map. The refined particles were further cleaned up with one round of ab initio reconstruction (K = 4) in cryoSPARC and 110,090 particles remained. Finally, these particles were subjected to the non-uniform refinement with C2 symmetry in cryoSPARC, which yielded the final map at 3.5 Å resolution.

For TACAN^{H196A H197A}, 10,541 super-resolution movie stacks were collected. Motion-correction, twofold binning to a pixel size of 0.515 Å, and dose weighting were performed using MotionCor2 (Zheng et al., 2017). CTF parameters were estimated with CTFFind4 (Rohou and Grigorieff, 2015). Micrographs with ice contamination were removed manually, resulting in 9,600 micrographs for further processing. A total of 1,474,917 particles were auto-picked using Relion 3.1 and windowed into 400  ×  400-pixel images, then binned two times and subjected to 2D classification, yielding 975,636 particles. The following image processing workflow is identical to TACAN^WT sample. Briefly, these particles were subjected to ab initio reconstruction in cryoSPARC-2.9.0 (Punjani et al., 2017), specifying four output classes. The best class with 607,159 particles was selected, then subjected to the resolution-based classification, yielding 391,137 particles. Subsequent non-uniform refinement with C2 symmetry in cryoSPARC was performed, resulting in a map with a resolution of 3.8 Å and the resolution was further improved to 3.3 Å by iterative Bayesian polishing and non-uniform refinement cycles. Particles were further cleaned up with one round of ab initio reconstruction with 155,946 particles remaining. Finally, these particles were subjected to the non-uniform refinement with C2 symmetry in cryoSPARC, which yielded the final map at 2.8 Å resolution.

The mask-corrected Fourier shell correlation (FSC) curves were calculated in cryoSPARC 2.9.0, and reported resolutions were based on the 0.143 criterion. Local resolutions of the final maps were estimated by Relion 3.1 (Scheres, 2020; Zivanov et al., 2020). A summary of reconstructions is shown in Table 1 and Figure 3—figure supplement 1E,F, Figure 3—figure supplement 2A,B, Figure 4—figure supplement 1B–E.

For TACAN^WT, the 3.5 Å resolution map was subjected to Buccaneer in the CCP-EM suite (Burnley et al., 2017; Wood et al., 2015) to generate the de novo model. This initial model was further improved using phenix.sequence_from_map in Phenix (Adams et al., 2010). Several iterative cycles of refinement using the phenix.real_space_refine with secondary structure and NCS restraints and manual adjustments in COOT yielded the final model for the TACAN^WT containing residues 9–72, 76-250 and 262–335 (Adams et al., 2010; Emsley et al., 2010).

For TACAN^{H196A H197A}, model of TACAN^WT was placed into the 2.8 Å map using UCSF Chimera (Pettersen et al., 2004) and manually adjusted in COOT Emsley et al., 2010 followed by iterative refinement cycles using the phenix.real_space_refine in Phenix with secondary structure and NCS restraints and manual adjustments in COOT. The final model for TACAN^{H196A H197A} contained residues 9–72, 76–250 and 262–335 as well as 2 CoASH molecules bound.

Refinement statistics are summarized in Table 1. Structural model validation was done using Phenix and MolProbity based on the FSC = 0.5 criterion (Chen et al., 2010). Figures were prepared using PyMOL (https://pymol.org/2/) and UCSF Chimera (Pettersen et al., 2004). Representative densities of TACAN^WT and TACAN^{H196A H197A} are shown in Figure 3—figure supplement 2C and Figure 4—figure supplement 1F, respectively.

The purified wild-type and mutant M. musculus TACAN samples were incubated with excess (0.7–0.9 mM) ligand (CoASH, S-ethyl-CoA, or acetyl-CoA) for 1 hr on ice. The incubated samples were then buffer exchanged into 200 mM ammonium acetate, 0.002% LMNG (2 CMC) using a Zeba microspin desalting column with a 40 kDa MWCO (Thermo Fisher Scientific). For nMS analysis, a 2–3 µL aliquot of each buffer-exchanged sample was loaded into a gold-coated quartz capillary tip that was prepared in-house and then electrosprayed into an Exactive Plus with extended mass range (EMR) instrument (Thermo Fisher Scientific) using a modified static direct infusion nanospray source (Olinares and Chait, 2020). The MS parameters used include spray voltage, 1.22–1.25 kV; capillary temperature, 125–200 °C; in-source dissociation, 125–150 V; S-lens RF level, 200; resolving power, 8,750 or 17,500 at m/z of 200; AGC target, 1 × 10⁶; maximum injection time, 200 ms; number of microscans, 5; injection flatapole, 8 V; interflatapole, 7 V; bent flatapole, 5 V; high-energy collision dissociation (HCD), 200 V; ultrahigh vacuum pressure, 6–7 × 10⁻¹⁰ mbar; and total number of scans, at least 100. The instrument was mass calibrated in positive EMR mode using cesium iodide.

For data processing, the acquired MS spectra were visualized using Thermo Xcalibur Qual Browser (v. 4.2.47). MS spectra deconvolution was performed either manually or with UniDec v. 4.2.0 (Marty et al., 2015; Reid et al., 2018). The UniDec parameters used included m/z range, 1500–5000; mass range, 20,000–100,000 Da; peak shape function, Gaussian; and smooth charge state distribution, on.

From their primary sequences, the expected masses for the proteins are TACAN^WT monomer: 41,770 Da, TACAN^WT dimer: 83,539 Da, TACAN^{H196A H197A} monomer: 41,637 Da, and TACAN^{H196A H197A} dimer: 83,275 Da. Experimental masses were determined as the average mass± standard deviation (SD) across all the calculated mass values in the relevant peak series (n ≥ 5 charge states) with typical SDs of ±1 Da.

Coenzyme A releasing activity was measured using a fluorescence-based coupled-enzyme assay (Sigma-Aldrich, Cat. MAK034) in a 96-well microplate (Costar) at 37°C. The reaction was monitored with an Infinite-M1000 spectrofluorometer (Tecan) with 535 nm excitation and 587 nm emission. The reconstituted proteoliposomes of ELOVL7 and TACAN at 1:50 protein-to-lipid ratio were used (10 µg total protein), supplemented with 100 µM malonyl-CoA (Sigma-Aldrich, Cat. M4263) and 50 µM stearoyl-CoA (Sigma-Aldrich, Cat. S0802). Reaction mixtures were incubated at 37°C for 0, 0.5, 1, 2, 4, 8, 24, and 48 hr, frozen in liquid N₂, and stored at –80°C. The mixtures were then centrifuged at 20,817 g for 10 min, and supernatants were used to perform the enzymatic assay following the manufacturer’s protocol.