Regulatory switch at the cytoplasmic interface controls TRPV channel gating

  1. Lejla Zubcevic
  2. William F Borschel
  3. Allen L Hsu
  4. Mario J Borgnia
  5. Seok-Yong Lee  Is a corresponding author
  1. Duke University School of Medicine, United States
  2. National Institute of Environmental Health Sciences, National Institutes of Health, United States
6 figures, 2 tables and 1 additional file

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, …

https://doi.org/10.7554/eLife.47746.002
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

https://doi.org/10.7554/eLife.47746.003
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) …

https://doi.org/10.7554/eLife.47746.005
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 …

https://doi.org/10.7554/eLife.47746.006
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 …

https://doi.org/10.7554/eLife.47746.007
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 …

https://doi.org/10.7554/eLife.47746.009
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 …

https://doi.org/10.7554/eLife.47746.010
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) …

https://doi.org/10.7554/eLife.47746.011
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 …

https://doi.org/10.7554/eLife.47746.014
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 …

https://doi.org/10.7554/eLife.47746.015
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 …

https://doi.org/10.7554/eLife.47746.016
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

https://doi.org/10.7554/eLife.47746.017
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 …

https://doi.org/10.7554/eLife.47746.018
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 …

https://doi.org/10.7554/eLife.47746.019
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 …

https://doi.org/10.7554/eLife.47746.020
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: …

https://doi.org/10.7554/eLife.47746.021
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, …

https://doi.org/10.7554/eLife.47746.023
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 …

https://doi.org/10.7554/eLife.47746.024
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 …

https://doi.org/10.7554/eLife.47746.026

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
Magnification130,000x75,000x
Voltage (kV)300300
Electron exposure (e–/Å2)4042
Defocus range (μm)1–2.51.25–3
Pixel size (Å)1.061.08
Symmetry imposedC4C4
Initial particle images (no.)452,3881,174,521
Final particle images (no.)95,18479,006
Map resolution (Å)4.13.6
FSC threshold0.1430.143
Refinement
Initial model used (PDB code)6MHO6MHO
Model resolution (Å)4.13.6
FSC threshold0.1430.143
Map sharpening B factor (Å2)−120−100
Model composition
Non-hydrogen atoms17,33217,800
Protein residues25002492
Ligands04 (2-APB)
B factors (Å2)
Protein87.4340.51
Ligandn/a35.66
R.m.s. deviations
Bond lengths (Å)0.0080.008
Bond angles (°)0.8680.833
MolProbity score1.641.24
Clashscore55
Poor rotamers (%)00
Ramachandran plot
Favored (%)92.7097.01
Allowed (%)7.302.99
 Disallowed (%)00
Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Cell line
(E. coli)
DH10BacThermoFisher Scientific10361012
Cell line
(Spodoptera frugiperda)
Sf9ATCCCRL-1711RRID:CVCL_0549
Cell line
(Homo sapiens)
HEK293TATCCCRL-11268; Lot
Number 62312975
RRID:CVCL_0063
Cell media
component
Dulbecco’s Modified
Eagle’s Medium (DMEM)
- low glucose
Gibco11885–084
Cell media
component
Heat Inactivated
Fetal Bovine Serum
Gibco10082–139
Cell media
component
Anti-Anti
(Antibiotic-Antiycotic)
Gibco15240–062
Recombinant
DNA reagent
human TRPV3GenscriptPubmed
Gene ID: 162514
Recombinant
DNA reagent
Bac-to-Bac
Baculovirus
Expression System
ThermoFisher Scientific10359016
Recombinant
DNA reagent
FuGene6PromegaE2691
Chemical
compound, drug
n-dodecyl-β-d-
maltopyranoside(DDM)
AnatraceD310
Chemical
compound, drug
Cholesteryl
Hemisuccinate
AnatraceCH210
Chemical
compound, drug
PMAL-C8AnatraceP5008
Chemical
compound, drug
TRISFisher ScientificBP152
Chemical
compound, drug
NaClFisher ScientificS271
Chemical
compound, drug
CaCl2Fisher ScientificC70
Chemical
compound, drug
KClSigma AldrichP9333
Chemical
compound, drug
MgCl2Sigma AldrichM8266
Chemical
compound, drug
4-(2-hydroxyethyl)−1-
piperazineethanesulfonic
acid (HEPES)
Sigma AldrichH3375
Chemical
compound, drug
NaOHSigma AldrichS5881
Chemical
compound, drug
CsClSigma AldrichC3139
Chemical
compound, drug
Ethylene glycol-bis
(2-aminoethylether)-
N,N,N′,N′-tetraacetic
acid (EGTA)
Sigma AldrichE4378
Chemical
compound, drug
CsOH solutionSigma Aldrich232041
Chemical
compound, drug
2-Aminoethyl
diphenylborinate
(2-APB)
Sigma AldrichD9754
Chemical
compound, drug
D-CamphorSigma AldrichW223018
Chemical
compound, drug
Dimethyl sulfoxide
(DMSO)
Sigma AldrichD2650
Chemical
compound, drug
leupeptinGoldBioL-010
Chemical
compound, drug
pepstatinGoldBioP-020
Chemical
compound, drug
aprotininGoldBioA-655
Chemical
compound, drug
DNase IGoldBioD-301
Chemical
compound, drug
β-mercapto ethanolSigma AldrichM3148
Chemical
compound, drug
PMSFSigma AldrichP7626
Chemical
compound, drug
anti-FLAG resinSigma AldrichA4596
Chemical
compound, drug
Bio-Beads SM-2BioRad152–8920
Chemical
compound, drug
1-palmitoyl-2-oleoyl
-sn-glycero-3-phosphocholine
(POPC)
Avanti Polar Lipids850457C
Chemical
compound, drug
1-palmitoyl-2-oleoyl
-sn-glycero-3-
phosphoethanolamine
(POPE)
Avanti Polar Lipids850757C
Chemical
compound, drug
1-palmitoyl-2-oleoyl
-sn-glycero-3-phospho-
(1'-rac-glycerol) (POPG)
Avanti Polar Lipids840457C
Software,
algorithm
MotionCor2Zheng et al., 2017http://msg.ucsf.edu/em/software/motioncor2.htmlRRID:SCR_016499
Software,
algorithm
GCTFZhang, 2016https://www.mrc-lmb.cam.ac.uk/kzhang/RRID:SCR_016500
Software,
algorithm
RELION 3.0Zivanov et al., 2018https://www2.mrc-lmb.cam.ac.uk/relion/RRID:SCR_016274
Software,
algorithm
CootEmsley and Cowtan, 2004https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/RRID:SCR_014222
Software,
algorithm
PhenixAdams et al., 2010http://phenix-online.org/RRID:SCR_014224
Software,
algorithm
MolprobityChen et al., 2010http://molprobity.biochem.duke.edu/index.phpRRID:SCR_014226
Software,
algorithm
UCSF ChimeraPettersen et al., 2004https://www.cgl.ucsf.edu/chimera/RRID:SCR_004097
Software,
algorithm
PymolShrödinger LLChttps://pymol.org/2/RRID:SCR_000305
Software,
algorithm
pClamp10Molecular DevicesRRID:SCR_011323
Software,
algorithm
OriginPro 2016OriginLab Corp.RRID:SCR_014212
Software,
algorithm
Microsoft Excel 2010MicrosoftRRID:SCR_016137
otherWhatman No. one
filter paper
Sigma AldrichWHA1001325
OtherUltrAuFoil R1.2/1.3
300-mesh grid
Electron Microscopy
Sciences
Q350AR13A
OtherCryo-electron microscopy
structure of the human
TRPV3 channel
Zubcevic et al., 2018aPDB ID 6MHOZubcevic et al., 2018b
OtherCryo-electron microscopy
structure of the human
TRPV3 channel
Zubcevic et al., 2018aEMDB ID EMD-9115Zubcevic et al., 2018b

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