Analysis of the mechanosensor channel functionality of TACAN

Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods, we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods, we found that TACAN, at high protein concentrations, produces heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single-particle cryo-electron microscopy and observed that it is a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme A cofactor, confirmed by mass spectrometry. The TACAN protomer is related in three-dimensional structure to a fatty acid elongase, ELOVL7. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.


Introduction
Mechanosensitive ion channels (MSCs) open in response to mechanical forces (Guharay and Sachs, 1984;Guharay and Sachs, 1985;Kung, 2005;Sachs, 2010). When the channels open, ions flow across the cell membrane, triggering subsequent biochemical processes that ultimately represent a cellular response to the applied mechanical force. This coupling of transmembrane (TM) ion flow to mechanical forces underlies some forms of osmoregulation, cell and organ growth, blood pressure regulation, touch, and hearing (Chalfie, 2009;Coste et al., 2010;Pan et al., 2013;Peyronnet et al., 2012;Woo et al., 2015). Several MSCs have been discovered and characterized (Kefauver et al., 2020). Recently, a new MSC in mammals called TACAN was reported and proposed to mediate mechanical pain (Beaulieu-Laroche et al., 2020). TACAN, originally identified in a proteomics screen and called TMEM120A, was categorized as a nuclear envelope protein (NET29) that participates in lipid metabolism (Batrakou et al., 2015;Byerly et al., 2010;Haakonsson et al., 2013;Lee et al., 2005;Rosell et al., 2014). Adipocyte-specific TMEM120A knockout mice exhibited a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2 (Czapiewski et al., 2021).
As our laboratory studies the biophysical mechanisms by which MSCs transduce mechanical forces and conduct ions across membranes, we were intrigued by TACAN's potential role as an MSC and set out to examine this function and report our findings here.  (GUVs). All recordings were performed with identical pipette and bath solution containing 10 mM HEPES pH 7.4, 140 mM KCl, and 1 mM MgCl 2 (~300 Osm/L). Traces were obtained holding at -80 mV with a pressure pulse protocol shown at the bottom: 0 to -80 mmHg with 10 mmHg step. Traces colored in red represent the observed currents with -80 mmHg pressure pulse.
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.

Structural analysis of TACAN
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 Å 2 , 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   The protein is colored rainbow from N-terminus (blue) to C-terminus (red). The six transmembrane helices (S1-S6), two horizontal helices (H1 and H2), as well as a short helix (H3) in between are labeled.
The online version of this article includes the following figure supplement(s) for figure 3:     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 3figure 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 5figure 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.

Discussion
We undertook this study to understand how TACAN functions as an MSC, but have been unable to replicate evidence of MSC activity. We observe no channel activity in the plasma membrane of cells expressing TACAN and the heterogeneous-in-amplitude currents (without mechanosensitive properties) that we observe when we reconstitute TACAN at high protein concentrations are not consistent with other native biological channels that we have studied.
Structurally, TACAN is related to coenzyme A-dependent FA elongases; however, without further data we cannot conclude that TACAN itself functions as an enzyme. It also remains to be determined which membranes in a cell express TACAN.
In conclusion, we do not find evidence that TACAN is a mechanosensitive ion channel. The strength of this conclusion is in the electrophysiological interrogation. The structure, because it looks like a known enzyme, is compatible with the 'not a channel' conclusion, but the structure alone would not make a strong argument. A number of ion channels, including CLC channels (Dutzler et al., 2002;Dutzler et al., 2003;Feng et al., 2012;Park et al., 2017;Park and MacKinnon, 2018), TMEM16 (Dang et al., 2017;Paulino et al., 2017), and CFTR Zhang et al., 2017;Zhang et al., 2018), are not obviously ion channels based on their structures and indeed each are fairly indistinguishable from proteins exhibiting non-channel functions.

Protein expression and purification
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 GnTlcells (ATCC, CRL-3022) grown in suspension at a density of ~3 × 10 6 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 N 2 , 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-Dmaltopyranoside (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.

Proteoliposome reconstitution
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 N 2 and stored at −80 °C.

GUV formation
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-1.5-14 C) 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 MgCl 2 ) 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.
Cell culture and transfection for patch recordings 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).

Excised inside-out patch recordings
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 highspeed 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 MgCl 2 (~300 Osm/L).

Cell lines
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.

Cryo-EM sample preparation and data collection
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/Å 2 /s, resulting in a total dose of about 75.4 e − /Å 2 (TACAN WT ), or a 1.5 s movie stack of 38 frames with a dose rate of 37.7 e/Å 2 /s, resulting in a total dose of about 56.6 e/Å 2 (TACAN H196A H197A ).

Image processing
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

Model building and refinement
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 . 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 4figure supplement 1F, respectively.
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.