Structural mechanism of heat-induced opening of a temperature-sensitive TRP channel.

Construct The full-length mTRPV3 (residues 1–825) used in cryo-EM studies was cloned into a pEG BacMam vector 50 , with a C-terminal thrombin cleavage site (LVPRG), followed by a streptavidin affinity tag (WSHPQFEK), as was done previously 12 , 27 .

Expression and purification

TRPV3 construct was expressed and purified as previously described for mTRPV3 12 , 27 with minor modifications. Bacmids and baculoviruses were produced using a standard method 50 . Briefly, baculovirus was made in Sf9 cells for ~72 hours (Thermo Fisher Scientific, mycoplasma test negative, GIBCO #12659017) and was added to the suspension adapted HEK 293 cells lacking N-acetyl-glucosaminyltransferase I (GnTI – , mycoplasma test negative, ATCC #CRL-3022) that were maintained in Freestyle 293 media (Gibco-Life Technologies #12338–018) supplemented with 2% FBS at 37°C and 5% CO 2 . Twenty-four hours after transduction, 10 mM sodium butyrate was added to enhance protein expression, and the temperature was reduced to 30°C. Seventy-two hours after transduction, the cells were harvested by centrifugation at 5,471 g for 15 min using a Sorvall Evolution RC centrifuge (Thermo Fisher Scientific), washed in phosphate buffer saline (PBS, pH 8.0), and pelleted by centrifugation at 3,202 g for 10 min using an Eppendorf 5810 centrifuge. The cell pellet was resuspended in ice-cold lysis buffer, containing 20 mM Tris (pH 8.0), 150 mM NaCl, 0.8 μM Aprotinin, 4.3 μM Leupeptin, 2 μM Pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM β-mercaptoethanol (βME). Cells were subsequently lysed using a Misonix Sonicator with a preset program (6 cycles of 15 s “on” at the amplitude of 8 followed by 15 s “off”; this program was repeated three times for optimal cell lysis) under constant stirring on ice. Unbroken cells and cell debris were pelleted using an Eppendorf 5810 centrifuge at 3,202 g and 4°C for 10 min. The supernatant was subjected to ultracentrifugation in a Beckman Coulter ultracentrifuge using a Beckman Coulter Type 45Ti rotor at 186,000 g and 4°C for 1 hour to pellet the membranes. The membrane pellet was mechanically homogenized and solubilized in the lysis buffer supplemented with 2% (w/v) digitonin under stirring at 4°C for 2 hours. Insoluble material was removed by ultracentrifugation for 40 min in a Beckman Coulter Type 45Ti rotor at 186,000 g, and the supernatant was added to strep resin and rotated for 14–16 hours at 4°C. Next, the resin was washed with 10 column volumes of buffer containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM βME, 0.01% (w/v) glyco-diosgenin (GDN) and the protein was eluted with the same buffer supplemented with 2.5 mM D-desthiobiotin. The eluted protein was concentrated using a 100 kDa NMWL centrifugal filter (MilliporeSigma ™ Amicon ™ ) to 0.5 ml and then centrifuged in a Sorvall MTX 150 Micro-Ultracentrifuge (Thermo Fisher Scientific) using a S100AT4 rotor for 30 min at 66,000 g and 4°C before injecting into a size-exclusion chromatography (SEC) column. The protein was further purified using a Superose ™ 6 10/300 GL SEC column attached to an AKTA FPLC (GE Healthcare) and equilibrated in 150 mM NaCl, 20 mM Tris, 1 mM βME, 0.01% GDN (pH 8.0). The tetrameric peak fractions were pooled and concentrated using 100 kDa NMWL centrifugal filter (MilliporeSigma ™ Amicon ™ ) to 2–4 mg/ml. For TRPV3 reconstitution into MSP2N2 nanodiscs, the purified protein was mixed with MSP2N2 and soybean lipids (Soy polar extract, Avanti Polar Lipids, USA) at a molar ratio of 1:3:166 (monomer:MSP2N2:lipid). The MSP2N2 was stored in a buffer containing 150 mM NaCl and 20 mM Tris (pH 8.0). The lipids were dissolved to a concentration of 100 mg/ml in 150 mM NaCl, 20 mM Tris (pH 8.0) and subjected to 5–10 cycles of freezing in liquid nitrogen and thawing in a water bath sonicator. The nanodisc mixture (500 μl) was rocked at room temperature for 1 hour. Subsequently, 20 mg of Bio-beads SM2 (Bio-Rad) pre-wet in buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM βME) was added to the nanodisc mixture and then it was rotated at 4°C. After one hour, additional 20 mg of Bio-beads SM2 was added and the resulting mixture was rotated at 4°C for ~14–20 hours. The Bio-beads SM2 were then removed by pipetting and mTRPV3 reconstituted in nanodiscs was purified from empty nanodiscs by SEC using Superose ™ 6 10/300 GL SEC column equilibrated in 150 mM NaCl, 20 mM Tris (pH 8.0), 1 mM βME. The fractions of mTRPV3 reconstituted into nanodiscs were pooled and concentrated using 100 kDa NMWL centrifugal filter (MilliporeSigma ™ Amicon ™ ) to 2–3 mg/ml and 2–4 mM EGTA was added to the sample. cNW11 circularized nanodiscs were prepared as described previously 28 and stored (~2–3 mg/ml) before usage at −80°C in 20 mM Tris (pH 8.0) and 150 mM NaCl. Purified mTRPV3 was incorporated into cNW11 circularized nanodiscs using the same procedure described above for the MSP2N2 nanodiscs.

Cryo-EM sample preparation and data collection

Au/Au grids were prepared as described in the literature 51 . Briefly, grids were prepared by first coating C-flat (Protochips, Inc., Morrisville, NC) CF-1.2/1.3–2Au mesh holey carbon grids with ~60 nm gold using an Edwards Auto 306 evaporator. Subsequently, an Ar/O 2 plasma treatment (4 min, 50 watts, 35.0 sccm Ar, 11.5 sccm O 2 ) was used to remove the carbon with a Gatan Solarus (model 950) Advanced Plasma Cleaning System (Gatan, Pleasanton, CA, USA). The grids were again plasma treated (H 2 /O 2 , 25 s, 10 watts, 6.4 sccm H 2 , 27.5 sccm O 2 ) prior to sample application to make their surfaces hydrophilic. These grids were used for MSP2N2-reconstituted specimens and specimens in detergent. For cNW11-reconstituted specimens, UltrAuFoil R 1.2/1.3, Au 300 grids were used. Prior to sample application and subsequent plunge-freezing, grids were plasma treated in a PELCO easiGlow glow discharge cleaning system (0.39 mBar, 15 mA, ‘glow’ 25 s, ‘hold’ 10 s). Before subjecting TRPV3 protein to cryo-EM sample preparation, it was tested in different heat application protocols followed by tetrameric peak detection using Fluorescence-detection Size-Exclusion Chromatography (FSEC) 52 , 53 . Only those protocols were used, during which the tetrameric peak amplitude was not significantly reduced. For heat stimulation cryo-EM experiments the samples were pre-incubated in T100 Thermal Cycler (Bio-Rad) programmed to generate repetitive constant temperature steps (30 s at 25°C and 30 s at 42°C). After incubating the sample for 10 to 20 min using repetitive temperature pulses, 3 μl of the sample was immediately transferred to a Mark IV Vitrobot (Thermo Fisher Scientific) equilibrated at 42°C and 100% humidity, applied to a goal-coated side of the grid, and plunge-freezing was done in liquid ethane using a blot time of 3–5 s, a blot force of 3–5 and a wait time of 30 s. The samples without heat stimulation were prepared using the method described above, except for the samples were pre-incubated for 30 min on ice before plunge-freezing, and the temperature inside the Vitrobot was set to 4°C. The grids were stored in liquid nitrogen before imaging. All images were collected on Titan Krios transmission electron microscopes (TEMs) (Thermo Fisher Scientific) operating at 300 kV and equipped with a post-column GIF Quantum energy filter and a Gatan K3 Summit direct electron detection (DED) camera (Gatan, Pleasanton, CA, USA). Images of frozen-hydrated particles of mTRPV3 in MSP2N2 nanodiscs (1.7 mg/ml) at 42°C were collected using Leginon 54 . Total number of 20,408 micrographs were collected in counting mode with an image pixel size of 0.858 Å and a defocus range of −0.8 to −2.0 μm. The total dose of ~58 e − Å −2 was attained by using a dose rate of ~16 e − pixel −1 s −1 across 50 frames for 2.5 s total exposure time. Images of frozen-hydrated particles of mTRPV3 in MSP2N2 nanodiscs (1.6 mg/ml) at 4°C were collected using SerialEM. Total number of 9,702 micrographs were collected in counting mode with an image pixel size of 0.826 Å and a defocus range of −0.8 to −2.0 μm. The total dose of ~58 e − Å −2 was attained by using a dose rate of ~11.6 e − pixel −1 s −1 across 50 frames for 2.5 s total exposure time. Images of frozen-hydrated particles of mTRPV3 in cNW11 nanodiscs (2.4–3.2 mg/ml) at 42°C were collected in three sessions using Leginon or EPU software (Thermo Fisher). Total number of 18,667 micrographs were collected in counting mode with an image pixel size of 0.87 or 0.873 Å and a defocus range of −0.5 to −2.0 μm, and energy filter slit of 20 eV. The total dose of ~58–60 e − Å −2 was attained by using a dose rate of ~16 e − pixel −1 s −1 across 50 frames for a 2.5 s total exposure time. Images of frozen-hydrated particles of mTRPV3 in cNW11 nanodiscs (2.4 mg/ml) at 4°C were collected using Leginon. Total number of 10,878 micrographs were collected in counting mode with an image pixel size of 1.083 Å and a defocus range of −0.8 to −2.0 μm, and energy filter slit of 20 eV. The total dose of 51.07 e − Å −2 was attained by using a dose rate of ~30 e − pixel −1 s −1 across 40 frames for a 2.0 s total exposure time.

Image processing

All processing were completed in RELION 55 and/or cryoSPARC 56 ( Table 1 ). The initial drift and beam-induced motion were corrected using MotionCor2 57 algorithm implemented in RELION 55 , and contrast transfer function (CTF) estimation was performed using Gctf 58 . Following CTF estimation, micrographs were manually inspected and those with outliers in defocus values, ice thickness, and astigmatism as well as micrographs with lower predicted CTF-correlated resolution (> 6 Å) were excluded from the rest of the processing pipeline (individually assessed for each parameter relative to overall distribution; no set threshold). Initial set of particles was picked using 4.48 Å cryo-EM map of mTRPV3 (EMD-20494) 12 for 3D reference-based picking in RELION and further classified into 100 two-dimensional (2D) classes. A selection of 2D classes was used to generate templates to be used for the final round of template-based picking. Picked particles were further 2D- and 3D-classified in iterative classification and selection rounds with 20 Å lowpass-filtered map EMD-20494 as a starting reference model using RELION 55 and/or cryoSPARC 56 . The reported resolutions of the final maps were estimated using the gold standard Fourier shell correlation in RELION 55 . The local resolution predictions were calculated in RELION, with the resolution range estimated by the gold standard FSC = 0.143 criterion 55 .

Map versus model

FSC curves with and without mask were calculated using Mtriage as part of Phenix package 59 . EM density visualization was done in UCSF Chimera 60 and UCSF ChimeraX 61 . As a representative of the image processing workflow, data for TRPV3 in cNW11 nanodiscs was processed as follows (see also Extended Data Fig. 6 ). Initially, 27119 particles were manually picked from 273 random picked micrographs in RELION 55 previously published 4.48 Å cryo-EM map of mTRPV3 (EMD-20494) 12 as s reference to generate 2D classes that were subsequently used as templates to automatically pick a total of 1,202,279, 2,886,493 and 2,780,712 particles from three separate collections. The extracted 4x-binned particle images were separately imported to cryoSPARC and subjected to several iterative rounds of 2D classification and heterogeneous refinement (3D classification). The particle images from the final 2D selections (131,394, 144,122 and 70,362, respectively) were imported separately back to RELION, re-extracted using the original box size of 256 pixels (without binning) and subjected to 3D auto-refinement with C1 symmetry using a box- and pixel-size corrected, previously published 4.48 Å cryo-EM map of mTRPV3 (EMD-20494) 12 as a template volume. The aligned particles were CTF refined to estimate beamtilt, trefoil, 4 th order aberrations, anisotropic magnification, and to fit per-particle defocus and per-micrograph astigmatism. The CTF-refined particles were further subjected to Bayesian polishing. Polished particles from three collections were joined and refined with the C1 symmetry to get a consensus 4.12-Å resolution map. Aligned particles from the previous step were subjected to 3D classification into eight classes with no symmetry imposed using the consensus map as a template. Particles from two best classes (36,446 and 39,172 in total) were refined with the C4 symmetry, with the respective map from last 3D classification step as a template. The particles form the class with an obvious closed pore was CTF-refined again as it was described previously and refined with the C4 symmetry using a soft mask covering the protein and nanodisc densities. The final post-processing with the soft mask resulted in a 3.09 Å map that represented the sensitized state of mTRPV3. The particles form the class with an obvious open pore was CTF-refined as was described previously and refined with the C4 symmetry using a soft mask covering the protein and nanodisc densities. To improve the quality of putative lipid densities, the refined map from the previous step was subjected to focussed 3D classification into four classes with a mask around transmembrane part of the protein. The total of 27,805 particles from the best class were refined with the C4 symmetry and postprocessed, which resulted in 3.48-Å resolution map. The final step of density-modification procedure 62 was used to further enhance the quality of putative lipid densities that resulted in 3.45 Å map of mTRPV3 in the open state. Model building To build models of TRPV3 in Coot 63 , we used the previously published cryo-EM structures of TRPV3 as guides 12 , 27 . The models were tested for overfitting by shifting their coordinates by 0.5 Å (using Shake) in Phenix 64 , refining each shaken model against a corresponding unfiltered half map, and generating densities from the resulting models in Chimera. Structures were visualized, and figures were prepared in UCSF Chimera 60 , UCSF ChimeraX 61 , and Pymol 65 .

MD simulations

Structural models of TRPV3 in the closed, sensitized and open states were immersed into a fully hydrated lipid bilayer with the molecular composition close to composition of HEK 293 cell membrane 66 : 25% palmitoyloleoylphosphatidylcholine (PC), 15% palmitoyloleoylphosphatidylethanolamine (PE), 15% palmitoyloleoylphosphatidylserine (PS), 15% palmitoyloleoylphosphatidylglycerol (PG), 5% 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol (PI), 10% sphingomyelin (SMP) and 30% cholesterol (about 870 molecules in total) using the in-house IMPULSE software. About 180 sodium ions were added for electroneutrality. Because of the gap between the residues 76 and 113 in the open-state structure, the 58–76 segments were treated as independent polypeptides with added N-acetyl and methylamide terminal groups. Six different lipids (PC, PE, PS, PG, PI and cholesterol) were inserted into the vanilloid site of the closed-state structure using the cryo-EM structure-modeled lipid as a template ( Fig. 4 ). The simulated systems were first equilibrated in several stages: 5 × 10 4 steps of steepest descent minimization followed by heating from 5 to 315 K during 100 ps MD run and 10 ns of MD run at 315 K with fixed positions of the protein atoms to permit membrane relaxation after insertion of the protein. Then, 500-ns MD production runs were carried out for each system. MD simulations were performed using the GROMACS 2020.4 package 67 , Amber99sd-ildn force field 68 , and TIP3P water model 69 . Simulations were carried out with an integration time of 2 fs, imposed 3D periodic boundary conditions, constant temperature (315 K) and pressure (1 bar). Electrostatic interactions were evaluated using the particle-mesh Ewald summation (real space cutoff 15 Å and 1.2 Å grid with fourth-order spline interpolation). A twin-range (15/15 Å) spherical cutoff function was employed to treat van der Waals interactions. Conductance of water and Na + ions through the selectivity filter and gate was calculated by explicitly accounting for water molecules (ions) consecutively passing through the two planes (in both directions) placed 7–8 Å above and below the residues G638 and I674 that form the narrow constrictions ( Fig. 2c – d ). Spatial distributions of water molecules and Na + in the pore and lipids at the vanilloid site were calculated as their densities averaged over the MD trajectory at the time range of 100–500 ns.

Whole-cell patch-clamp recordings

Whole-cell patch-clamp recording were parformed as described previously 45 . HEK293T cells were cultured in Opti-MEM I medium (Invitrogen) supplemented with 5% fetal bovine serum (PAN-Biotech, Germany). The day before transfection, cells were plated in 24-well plates (2 × 10 5 cells per well) in 0.5 ml of medium and became confluent on the day of transfection. The cells were transiently co-transfected with 300 ng of plasmid encoding wild-type mouse TRPV3 (in pcDNA3 vector, kind gift of Prof. Michael Caterina, Johns Hopkins University School of Medicine, Baltimore, MD, USA), and with 200 ng of GFP plasmid (Takara, Japan) using the magnet-assisted transfection technique (IBA GmbH, Goettingen, Germany) and then plated on poly-L-lysine-coated glass coverslips. The presented data are representative of comparable results from 19 experiments with 6 independent transfections. The cells were used 24 h after transfection. Whole-cell membrane currents were recorded by employing an Axopatch 200B amplifier and pCLAMP 10.2 software (Molecular Devices, Sunnyvale, USA). Patch electrodes were pulled from borosilicate glass capillary with a 1.5-mm outer diameter (Science Products GmbH, Germany). The tip of the pipette was heat-polished, and its resistance was 3–6 MΩ. Series resistance was compensated by 50–70% after compensation of fast and slow capacitance. Only one recording was performed on any one coverslip of cells to ensure that recordings were made from cells not previously exposed to heat. The extracellular control bath solution for recording contained: 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 320 mOsm, adjusted to pH 7.4 with NaOH. The pipette solution contained 150 mM CsCl, 1 MgCl 2 , 10 HEPES and 5 mM EGTA (295 mOsm, pH 7.4 adjusted with CsOH). A system for rapid superfusion of the cultured cells was used for thermal stimulation and solution application 70 . Briefly, experimental solutions were driven by gravity from seven different barrels, through automatically controlled valves, to a manifold that consisted of fused silica tubes connected to a common outlet glass capillary. The lower part of the capillary was wrapped with densely coiled copper wire that heated the solution to a chosen final temperature. The volume of solution in the experimental dish and the immersion of the application capillary were maintained at a constant level and an internal table generated after balancing an electrical circuit of the system was utilized, which resulted in a good reproducibility of the heat stimuli. The average speed of temperature changes did not significantly differ throughout all experiments. Voltage commands for heat were generated from Digidata 1440A digitizer using pCLAMP 10.2 software (Molecular Devices, Sunnyvale, CA, USA). Temperature steps from room temperature to > 50°C with pulse duration of 5 s were applied at 5 or 10-s intervals. All chemicals were purchased from Sigma-Aldrich (Merck, Prague, Czech Republic).

Planar lipid bilayer recordings

Planar lipid bilayers measurements were performed as described previously 71 . Briefly, planar lipid bilayers were formed from a solution of synthetic 1-palmitoyl-2-oleoyl-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine (POPE; Avanti Polar Lipids) at a 3:1 ratio in n -decane (Sigma-Aldrich). The solution was used to paint a bilayer in an aperture of ~250 μm diameter in a Delrin cup (Warner Instruments) between symmetric aqueous bathing solutions of 150 mM KCl, 0.02 mM MgCl 2 , and 20 mM HEPES (pH 7.2), in the presence of 4 mM EGTA. All reagents (Sigma-Aldrich) were ultrapure (>99%). Bilayer capacitances were in the range of 50–75 pF. After the bilayers had formed, the TRPV3 protein was added by painting from the micellar solution of TRPV3 protein (20 ng/ml). Unitary currents were recorded using the Axopatch 200B patch-clamp amplifier (Molecular Devices). The trans solution (command voltage side) was connected to the CV 201A head-stage input, while the cis solution was held at a virtual ground via a pair of matched Ag-AgCl electrodes. Currents through the voltage-clamped bilayers (background conductance,