Regulatory switch at the cytoplasmic interface controls TRPV channel gating
- Duke University School of Medicine, United States
- National Institute of Environmental Health Sciences, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
The role of the cytoplasmic inter-protomer interface in hTRPV3 gating.
(A) Architecture of the hTRPV3 protomer. Ankyrin repeat domain (ARD) is colored in cyan, the coupling domain (CD) and its individual elements (HLHCD, βCD, Pre-S1CD) are colored in blue, …
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Figure 1—source data 1
This spreadsheet contains data points for the time course of 2-APB use-dependence plots in Figure 1D, 1F and 1H; current density data used to generate bar plots in Figure 1I; initial sensitization data used to generate bar plots in Figure 1J.
- https://doi.org/10.7554/eLife.47746.004
Figure 1—figure supplement 1
State model and gating schemes of TRPV3 channels.
Simple state model of TRPV3 gating proposed by Liu et al. (2011). The transition rate from C1 → C0 (scheme 1) and O→C0 (scheme 2) is slow enough that this transition is irreversible. Both k0 and k1 …
Figure 2 with 2 supplements
Rearrangements of the cytoplasmic domains in the TRPV3K169A structure.
(A) The cytoplasmic inter-protomer interface in TRPV3WT (left panel) and TRPV3K169A (right panel). The CTD and the putative N-terminal region are highlighted in red and purple, respectively. (B) …
Figure 2—figure supplement 1
Cryo-EM data collection and processing, TRPV3K169A.
(A) A representative micrograph from the TRPV3K169A data collection. (B) 3D reconstruction workflow. (C) Euler distribution plot. Red regions show the best represented views. (D) Local resolution …
Figure 2—figure supplement 2
Electron density in the TRPV3K169A cryo-EM map.
(A) Representative electron densities in the TRPV3K169A cryo-EM map. Densities are contoured at level 0.015 and radius 2. (B–C) Connectivity between the ARD and the putative N-terminal region. The …
Figure 3 with 2 supplements
State-dependent changes at the cytoplasmic inter-protomer interface.
(A–B) Top view of the cytoplasmic inter-protomer interactions in TRPV3WT (A) and TRPV3K169A (B). In TRPV3WT the CTD (red) coils around the βCD (grey). The distal CTD interacts with the ARD at the …
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Figure 3—source data 1
This spreadsheet contains current density data used to generate bar plots in Figure 3H; initial sensitization data used to generate bar plots in Figure 3I; calculated dose-response values used to generate bar plots in Figure 3J.
- https://doi.org/10.7554/eLife.47746.013
Figure 3—figure supplement 1
Conformational changes in the cytoplasmic domains in TRPV3K169A.
(A) Overlay of the tetrameric cytoplasmic assemblies from TRPV3WT (orange) and TRPV3K169A (blue). Close-up view single ARD shows that the tetrameric assemblies cannot be superposed through a rigid …
Figure 3—figure supplement 2
The TRPV3 W742A and K743A CTD mutants alter hysteresis.
Three consecutive rounds of 2-APB dose-responses (0 (black) 3(purple), 10 (blue), 30 (green), 50 (yellow), 100 (orange), 300 (red) μM) was applied for 15 s followed 15 s of wash for WT (A) W742A (B) …
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Figure 3—figure supplement 2—source data 1
This spreadsheet contains dose-response data used to calculate mean values plotted in Figure 3—figure supplement 2D.
- https://doi.org/10.7554/eLife.47746.012
Figure 4 with 7 supplements
The structure of TRPV3K169A 2-APB exhibits changes in both transmembrane and cytoplasmic domains.
(A) One 2-APB molecule is bound to each protomer of the TRPV3K169A 2-APB channel (magenta). 2-APB is found between the HLHCD, Pre-S1CD and TRP domains in a binding site defined by residues H417 in …
Figure 4—figure supplement 1
Cryo-EM data collection and processing, TRPV3K169A 2-APB.
(A) A representative micrograph from TRPV3K169A 2-APB data collection. (B) 3D reconstruction workflow. (C) Euler distribution plot. Red regions show the most represented views. (D) Local resolution …
Figure 4—figure supplement 2
Electron density in the TRPV3K169A 2-APB cryo-EM map.
Representative electron densities in the TRPV3K169A 2-APB cryo-EM map. Densities are contoured at level 0.03 and radius 2. The inset in the S6 panel shows a close-up of the density around the …
Figure 4—figure supplement 3
Comparison of the pore conformations of hTRPV3WT, hTRPV3K169A 2-APB and mTRPV3Open.
Bottom-up view of the hTRPV3WT (PDB ID 6MHO, orange), hTRPV3K169A 2-APB (magenta) and mTRPV3Open (PDB ID 6DVZ, green) pores. The conformation of hTRPV3K169A 2-APB resembles that of the mTRPV3Open …
Figure 4—figure supplement 4
Coupling between the cytoplasmic and transmembrane domains.
(A) Top view of the coupling between the TRP domain (light blue), the CD (orange, Pre-S1CD and HLHCD) and the AR5 (magenta) in TRPV3WT. (B) In the TRPV3K169A 2-APB structure, the coil-to-helix …
Figure 4—figure supplement 5
The CTD in thermoTRPV structures.
(A) The cryo-EM map (EMD-8921) and atomic structure of the open mTRPV3 (PDB 6DVZ). Close-up view shows the fit of the CTD region into the electron density. (B) hTRPV3K169A 2-APB fit into the cryo-EM …
Figure 4—figure supplement 6
APB binding in TRPV3K169A 2APB.
(A) Non-assigned densities (red) in the VSLD cavity of TRPV3K169A (green, left) and TRPV3K169A 2-APB (purple, right). Electron densities are contoured at levels 0.015 and 0.03, respectively. (B) No …
Figure 4—figure supplement 7
APB and camphor response of proposed 2-APB binding site mutants.
(A) Graphical representation of the current response ratio of sub- (30 μM) and saturating 2-APB (300 μM) to camphor (10 mM) calculated as the mean from each biologically independent experiment. (WT: …
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Figure 4—figure supplement 7—source data 1
This spreadsheet contains data used to calculate the mean 2-APB to camphor ratio values plotted in Figure 4—figure supplement 7A.
- https://doi.org/10.7554/eLife.47746.022
Figure 5 with 1 supplement
Parallels between TRPV3K169A and wild-type TRPV1.
(A) Representative whole-cell recording at +60 mV of WT (black), K169A (red), E751A (blue), W739A (orange) immediately following 2-APB sensitization protocol, with perfusion protocol of 5 s wash, …
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Figure 5—source data 1
This spreadsheet contains the data used to calculate the ruthenium red sensitive current plots in Figure 5B and 5D; voltage step data used to calculate conductance plot in Figure 5E.
- https://doi.org/10.7554/eLife.47746.025
Figure 5—figure supplement 1
Sequence alignment of rat TRPV1 (rTRPV), rabbit TRPV2 (rabTRPV2), human TRPV3 (hTRPV3) and xenopus TRPV4 (xTRPV4).
The alignment is colored by conservation and regions of interest are marked with purple lines. residues of interest are indicated with *. The distal CTD has a high helical propensity as calculated …
Figure 6
The CTD-mediated ‘switch’ gating mechanism.
In the naive closed state (C0), the distal CTD is stretched around the βCD via a salt bridge ‘hook’ interaction with the ARD and the switch is ‘off’. When the CTD is ‘unhooked’ from the ARD, it …
Tables
Table 1
Cryo-EM data collection, refinement and validation statistics
https://doi.org/10.7554/eLife.47746.008| TRPV3K169A (EMD-20192) (PDB 6OT2) | TRPV3K169A 2-APB (EMD-20194) (PDB 6OT5) | |
|---|---|---|
| Data collection and processing | ||
| Magnification | 130,000x | 75,000x |
| Voltage (kV) | 300 | 300 |
| Electron exposure (e–/Å2) | 40 | 42 |
| Defocus range (μm) | 1–2.5 | 1.25–3 |
| Pixel size (Å) | 1.06 | 1.08 |
| Symmetry imposed | C4 | C4 |
| Initial particle images (no.) | 452,388 | 1,174,521 |
| Final particle images (no.) | 95,184 | 79,006 |
| Map resolution (Å) | 4.1 | 3.6 |
| FSC threshold | 0.143 | 0.143 |
| Refinement | ||
| Initial model used (PDB code) | 6MHO | 6MHO |
| Model resolution (Å) | 4.1 | 3.6 |
| FSC threshold | 0.143 | 0.143 |
| Map sharpening B factor (Å2) | −120 | −100 |
| Model composition | ||
| Non-hydrogen atoms | 17,332 | 17,800 |
| Protein residues | 2500 | 2492 |
| Ligands | 0 | 4 (2-APB) |
| B factors (Å2) | ||
| Protein | 87.43 | 40.51 |
| Ligand | n/a | 35.66 |
| R.m.s. deviations | ||
| Bond lengths (Å) | 0.008 | 0.008 |
| Bond angles (°) | 0.868 | 0.833 |
| MolProbity score | 1.64 | 1.24 |
| Clashscore | 5 | 5 |
| Poor rotamers (%) | 0 | 0 |
| Ramachandran plot | ||
| Favored (%) | 92.70 | 97.01 |
| Allowed (%) | 7.30 | 2.99 |
| Disallowed (%) | 0 | 0 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (E. coli) | DH10Bac | ThermoFisher Scientific | 10361012 | |
| Cell line (Spodoptera frugiperda) | Sf9 | ATCC | CRL-1711 | RRID:CVCL_0549 |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-11268; Lot Number 62312975 | RRID:CVCL_0063 |
| Cell media component | Dulbecco’s Modified Eagle’s Medium (DMEM) - low glucose | Gibco | 11885–084 | |
| Cell media component | Heat Inactivated Fetal Bovine Serum | Gibco | 10082–139 | |
| Cell media component | Anti-Anti (Antibiotic-Antiycotic) | Gibco | 15240–062 | |
| Recombinant DNA reagent | human TRPV3 | Genscript | Pubmed Gene ID: 162514 | |
| Recombinant DNA reagent | Bac-to-Bac Baculovirus Expression System | ThermoFisher Scientific | 10359016 | |
| Recombinant DNA reagent | FuGene6 | Promega | E2691 | |
| Chemical compound, drug | n-dodecyl-β-d- maltopyranoside(DDM) | Anatrace | D310 | |
| Chemical compound, drug | Cholesteryl Hemisuccinate | Anatrace | CH210 | |
| Chemical compound, drug | PMAL-C8 | Anatrace | P5008 | |
| Chemical compound, drug | TRIS | Fisher Scientific | BP152 | |
| Chemical compound, drug | NaCl | Fisher Scientific | S271 | |
| Chemical compound, drug | CaCl2 | Fisher Scientific | C70 | |
| Chemical compound, drug | KCl | Sigma Aldrich | P9333 | |
| Chemical compound, drug | MgCl2 | Sigma Aldrich | M8266 | |
| Chemical compound, drug | 4-(2-hydroxyethyl)−1- piperazineethanesulfonic acid (HEPES) | Sigma Aldrich | H3375 | |
| Chemical compound, drug | NaOH | Sigma Aldrich | S5881 | |
| Chemical compound, drug | CsCl | Sigma Aldrich | C3139 | |
| Chemical compound, drug | Ethylene glycol-bis (2-aminoethylether)- N,N,N′,N′-tetraacetic acid (EGTA) | Sigma Aldrich | E4378 | |
| Chemical compound, drug | CsOH solution | Sigma Aldrich | 232041 | |
| Chemical compound, drug | 2-Aminoethyl diphenylborinate (2-APB) | Sigma Aldrich | D9754 | |
| Chemical compound, drug | D-Camphor | Sigma Aldrich | W223018 | |
| Chemical compound, drug | Dimethyl sulfoxide (DMSO) | Sigma Aldrich | D2650 | |
| Chemical compound, drug | leupeptin | GoldBio | L-010 | |
| Chemical compound, drug | pepstatin | GoldBio | P-020 | |
| Chemical compound, drug | aprotinin | GoldBio | A-655 | |
| Chemical compound, drug | DNase I | GoldBio | D-301 | |
| Chemical compound, drug | β-mercapto ethanol | Sigma Aldrich | M3148 | |
| Chemical compound, drug | PMSF | Sigma Aldrich | P7626 | |
| Chemical compound, drug | anti-FLAG resin | Sigma Aldrich | A4596 | |
| Chemical compound, drug | Bio-Beads SM-2 | BioRad | 152–8920 | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphocholine (POPC) | Avanti Polar Lipids | 850457C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl -sn-glycero-3- phosphoethanolamine (POPE) | Avanti Polar Lipids | 850757C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl -sn-glycero-3-phospho- (1'-rac-glycerol) (POPG) | Avanti Polar Lipids | 840457C | |
| Software, algorithm | MotionCor2 | Zheng et al., 2017 | http://msg.ucsf.edu/em/software/motioncor2.html | RRID:SCR_016499 |
| Software, algorithm | GCTF | Zhang, 2016 | https://www.mrc-lmb.cam.ac.uk/kzhang/ | RRID:SCR_016500 |
| Software, algorithm | RELION 3.0 | Zivanov et al., 2018 | https://www2.mrc-lmb.cam.ac.uk/relion/ | RRID:SCR_016274 |
| Software, algorithm | Coot | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | RRID:SCR_014222 |
| Software, algorithm | Phenix | Adams et al., 2010 | http://phenix-online.org/ | RRID:SCR_014224 |
| Software, algorithm | Molprobity | Chen et al., 2010 | http://molprobity.biochem.duke.edu/index.php | RRID:SCR_014226 |
| Software, algorithm | UCSF Chimera | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | RRID:SCR_004097 |
| Software, algorithm | Pymol | Shrödinger LLC | https://pymol.org/2/ | RRID:SCR_000305 |
| Software, algorithm | pClamp10 | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | OriginPro 2016 | OriginLab Corp. | RRID:SCR_014212 | |
| Software, algorithm | Microsoft Excel 2010 | Microsoft | RRID:SCR_016137 | |
| other | Whatman No. one filter paper | Sigma Aldrich | WHA1001325 | |
| Other | UltrAuFoil R1.2/1.3 300-mesh grid | Electron Microscopy Sciences | Q350AR13A | |
| Other | Cryo-electron microscopy structure of the human TRPV3 channel | Zubcevic et al., 2018a | PDB ID 6MHO | Zubcevic et al., 2018b |
| Other | Cryo-electron microscopy structure of the human TRPV3 channel | Zubcevic et al., 2018a | EMDB ID EMD-9115 | Zubcevic et al., 2018b |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.47746.027
