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

Abstract

Numerous physiological functions rely on distinguishing temperature through temperature-sensitive transient receptor potential channels (thermo-TRPs). Although the function of thermo-TRPs has been studied extensively, structural determination of their heat- and cold-activated states has remained a challenge. Here, we present cryo-EM structures of the nanodisc-reconstituted wild-type mouse TRPV3 in three distinct conformations: closed, heat-activated sensitized and open states. The heat-induced transformations of TRPV3 are accompanied by changes in the secondary structure of the S2-S3 linker and the N and C termini and represent a conformational wave that links these parts of the protein to a lipid occupying the vanilloid binding site. State-dependent differences in the behavior of bound lipids suggest their active role in thermo-TRP temperature-dependent gating. Our structural data, supported by physiological recordings and molecular dynamics simulations, provide an insight for understanding the molecular mechanism of temperature sensing.

Extended Data Fig. 1. Temperature-dependent changes in currents and thermodynamics of TRPV3.

a, A representative continuous recording of current from multiple TRPV3 channels occasionally reconstituted into the synthetic lipid bilayer - (black) in response to the temperature ramp from 22 to 42°C (red), with the membrane potential alternating between +100 mV (red circles) and −100 mV (blue circles). Note the sharp increase in current activity in the 36–42°C temperature range. b, Temperature dependence of the open probability P_o at +100 mV calculated using single-channel recordings (see examples in Fig. 1b; n = 19 independent experiments; 189,976 events were analyzed; the data includes the previously published and new recordings). Fitting of the data allows estimation of the temperature coefficient, Q₁₀ = 27.0 ± 7.4 (n = 19 independent experiments). c, Van’t Hoff plot of the equilibrium constant K_eq calculated using the values of P_o (see Methods). Linear fits in the 22–36°C and 36–42°C temperature ranges provide the values of changes in enthalpy and entropy for the temperature-induced activation of TRPV3. Data in b and c are presented as mean values ± SEM. Source data for b and c are available online.

Extended Data Fig. 2. Characteristics of TRPV3 cryo-EM reconstructions.

Plots show unmasked, masked and corrected FSC curves calculated between half maps, with the overall resolution estimated using the FSC = 0.143 criterion. Cryo-EM maps are colored according to the local resolution estimation in Relion.

Extended Data Fig. 3. Map versus model FSC curves.

Map versus model FSC curves with and without mask were calculated using Mtriage as part of Phenix package.

Extended Data Fig. 4. Cryo-EM density of TRPV3.

a, Stereo view of an ARD fragment of the 1.98-Å resolution cryo-EM map of TRPV3 reconstituted in MSP2N2 nanodiscs and incubated at 4°C. b, Fragments of the same map for the membrane segments and TRP helix.

Extended Data Fig. 5. Comparison of pore geometry and architecture of TRPV3 structures.

a, Pore-forming domains of TRPV3 in the sensitized state with the residues lining the pore shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profile is shown as a space-filling model (light blue). b, Distribution of water (blue mesh) and ions (red mesh) during MD simulations of TRPV3 in the sensitized state, illustrating the lack of pore permeation. c, The pore radius for different TRPV3 structures calculated using HOLE. The vertical dashed line denotes the radius of a water molecule, 1.4 Å. d, Superposition of a single subunit from different closed-state structures of TRPV3, with the main structural elements labelled. The root-mean-square deviation (RMSD) calculated for each pair of these subunits ranges between 0.314 and 0.441 Å. e-f, Superposition of TRPV3 structures in the closed (MSP2N2, 4°C; green) and sensitized (pink) states (RMSD, 3.098 Å) viewed parallel to the membrane (e) or intracellularly (f). g-h, Superposition of TRPV3 structures in the closed (MSP2N2, 4°C; green) and open (orange) states (RMSD, 3.293 Å) viewed parallel to the membrane (g) or intracellularly (h). Only two of four subunits are shown in (e) and (g) with the front and back subunits omitted for clarity. Note (e-h) that TRPV3 in the sensitized and open states becomes shorter and its intracellular skirt undergoes a clockwise rotation when viewed intracellularly.

Extended Data Fig. 6. Overview of cryo-EM data collected for mTRPV3 in cNW11 nanodiscs at 42°C and 3D reconstruction workflow.

Representative micrographs with example particles circled in yellow and reference-free 2D class averages in different orientations are shown. Three datasets were collected and joined after particle clean-up. All processing steps were done in Relion, except the 2D/3D particle clean-up that was done in cryoSPARC.

Extended Data Fig. 7. Molecular dynamics simulations.

a, Conductance of water and Na⁺ ions through the selectivity filter and gate of the closed, sensitized and open TRPV3 plotted against the time course of MD simulation. Note that the closed state shows no permeation, the sensitized state permeates water through the selectivity filter only and the open state permeates water and Na⁺ ions through both selectivity filter and gate. b-g, Averaged MD density distributions (yellow) for non-hydrogen atoms of phosphatidylethanolamine (PE, b), phosphatidylinositol (PI, c), phosphatidylserine (PS, d), phosphatidylcholine (PC, e), phosphatidylglycerol (PG, f) and cholesterol (g) lipids nested in the vanilloid site of the closed TRPV3 at the beginning of 500-ns simulations. Black mesh shows cryo-EM density. MD snapshots of lipid molecules and residues coordinating their heads are shown in sticks. Chemical structures of the lipid molecules are shown to the left of each structural panel. The overlap of MD and EM densities are 26% for PE, 30% for PI, 36% for PS, 30% for PC, 33% for PG and 24% for cholesterol, calculated by multiplying the overlapping volume of MD and cryo-EM densities by two and dividing by their sum.

Extended Data Fig. 8. Comparison of open-state structures of wild-type TRPV3 and Y564A mutant.

a-b, Overall superposition (RMSD, 2.131 Å) of the open-state structures of wild-type TRPV3 (orange) and previously published Y564A mutant (blue, PDB ID: 6PVP) viewed parallel to the membrane (a) and extracellularly (b). c, Single-subunit superposition based on the transmembrane domains (RMSD, 0.943 Å). Note, the most pronounced conformational differences are observed for the S1-S2, S2-S3, S5-P and ARD loops, while the transmembrane domains and TRP helices superpose closely.

Extended Data Fig. 9. Sequence alignment of mouse TRPV channels.

α helices and β strands are depicted above the sequences as cylinders and arrows, respectively. The * symbols indicate residues in the ARD and linker domain that interact with residues in the C-terminus (¥ symbols). Red rectangular outlines denote regions involved in the interaction of the C-terminus with the ARD and linker domain, including residues conserved in thermo-TRPVs, and the AR5 and linker domain loops, which are present in thermo-TRPVs and absent in TRPV5-6. The location of the selectivity filter (S.F.) is indicated by a red box. Identical residues are colored red and highlighted in light pink. Positions of the previously identified mutations in TRPV3 that are critical for thermal sensitivity are highlighted in dark pink.

Extended Data Fig. 10. Conformational changes accompanying temperature-induced opening of wild-type TRPV3.

Superposition of the closed- and heat-activated open-state structures of TRPV3 (cNW11, 42°C) viewed parallel to the membrane is shown in the centre. Insets show select regions with the arrows indicating the displacement of domains in the open relative to the closed state. The lipid at the vanilloid site is shown in sticks (pink).

Figure 1.. TRPV3 function and cryo-EM.

a, Whole-cell patch-clamp current (black) recorded from HEK-293T cell expressing wild-type mouse TRPV3 in response to repetitive applications of heat (red) at −70 mV membrane potential. The dashed line indicates zero current. b, Representative single-channel currents recorded at 25°C, 36°C and 42°C and 30 mV membrane potential from wild-type TRPV3 reconstituted into lipid bilayers. The single-channel conductance measured at 42°C, 146 ± 7 (n = 15), is in the range of conductances measured in experiments with cells (48 – 261 pS, see Methods). The dotted lines indicate zero current. c, 1.98-Å resolution cryo-EM map of wild-type TRPV3 reconstituted in MSP2N2 nanodiscs and incubated at 4°C. d, Close-up view of the map region indicated by red rectangle in c. Protein is shown in sticks. Red spheres represent water molecules.

Figure 2.. TRPV3 structures and pore permeation at high temperature.

a-b, Closed-state (a) and open-state (b) structures of wild-type TRPV3 reconstituted in cNW11 nanodiscs and exposed to repetitive applications of heat. c-d, Pore-forming domains in the closed (c) and open (d) states with the residues lining the pore shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profiles are shown as space-filling models (light blue). e-f, Distribution of water (blue mesh) and ions (red mesh) during MD simulations of TRPV3 in the closed (e) and open (f) states, illustrating the lack and presence of pore permeation, respectively.

Figure 3.. N- and C-termini.

Interface between the neighbouring TRPV3 subunits (light pink and light blue) that connects elements of the intracellular skirt in the closed (a,c) and open (b,d) states, shown as cartoon (a-b) or surface (c-d), with the N-terminus shown in blue and C-terminus in hot pink and residues in stick representation.

Figure 4.. Lipids.

a-b, Cryo-EM density for cNW11-reconstituted wild-type TRPV3 in the closed (a) and heat-activated open (b) states, with the lipid densities coloured pink or blue. c-d, Close-up view of the membrane region, with the molecules of lipid shown in sticks and numbered. e-f, Close-up view of the vanilloid site (V), which harbours a phosphatidylcholine lipid in the closed state (sticks, e) and disappears in the open state (asterisk, f).

Figure 5.. State-dependent structural changes.

Light blue (no changes) to red (strong changes) gradient of RMSD (a-b) or translation (c-d) calculated between closed and sensitized (a, c) or sensitized and open (b, d) states and mapped on the sensitized state structure. RMSD values were calculated for Cα atoms along the entire TRPV3 sequence with a sliding window of 10 residues. Cα atom translations were calculated after aligning S1-S4 domains of the corresponding structures. Regions of the greatest structural changes are labelled. Blue arrows indicate the direction of the conformational wave accompanying closed to sensitized (c) and sensitized to open (d) state transitions.

Figure 6.. Mechanism of TRPV3 temperature activation.

Heat-induced activation of TRPV3 occurs in two steps: sensitization and channel opening. Sensitization is highly temperature-sensitive and accompanied by withdrawal of the vanilloid-site lipid and a massive conformational wave that includes the S2-S3 loop, N- and C-termini changing their secondary structures and positioning. As a result of sensitization, the intracellular skirt of TRPV3 rotates by ~8° and moves towards the transmembrane domain. Channel opening is weakly temperature-sensitive and accompanied by more local structural changes that involve the TRP helix and pore-forming S6 and P-loop and results in pore widening sufficient for ion conductance.