New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket
- Department of Physiology and Membrane Biology, University of California Davis, School of Medicine, United States
- Department of Pharmacology, University of California Davis, School of Medicine, United States
Figures
Figure 1
Novel capsaicin analogs for the study of TRPV1 ligand-binding pocket.
(A–C) Characteristics of the ligand-binding pocket with distinct vanilloid binding poses. (D) Distance between the two capsaicin hydrogen bonding residues T551 and E571 in the apo state (blue, PDB: …
Figure 2
Capsaicin analogs with a shorter or longer neck can fully activate TRPV1.
(A) Representative current traces induced by capsaicin or its analogs recorded at +80 mV (black) and −80 mV (red). (B) Concentration-response curves (n = 5–8 cells). (C) Representative …
Figure 3 with 3 supplements
Docking of Cap-1 (A–C) and Cap+1 (D–F) reveals interacting channel residues and binding poses of agonists.
(A) Representative binding poses of CAP-1 inside the ligand-binding pocket. (B) Energy of predicted VDW and hydrogen bond for each ligand-binding pocket residues. (C) Concentration-response curves …
Figure 3—figure supplement 1
Representatives of the vertical binding poses for Cap-1 (left) and the horizontal binding poses for Cap+1 (right) among the 30 top models.
Figure 3—figure supplement 2
Representative whole-cell patch-clamp recordings of TRPV1 T671S mutant activated by capsaicin (top left), Cap-1 (top right), and Cap+1 (bottom left), and the concentration-response curves (bottom right).
Figure 3—figure supplement 3
Representative whole-cell patch-clamp recordings of TRPV1 Y512F mutant activated by capsaicin (top left) and Cap-1 (top right), and the concentration-response curves (bottom).
Figure 4 with 3 supplements
Modifying the neck of capsaicin lowers the binding affinity and allosteric constant for gating.
(A) Comparison of the OFF rates. (B) Concentration-response curves of capsazepine inhibition, with a Hill function fit superimposed. n = 3–5 cells. (C) Gating scheme of capsaicin ligand binding to …
Figure 4—figure supplement 1
Capsaicin analogs exhibited a more rapid off rate.
Representative normalized tail current traces of TRPV1. Dotted curves represent a single-exponential fit to the tail currents.
Figure 4—figure supplement 2
Capsaicin analogs display a reduced binding affinity.
Current traces of capsazepine (CZP) competitively antagonized channel activation induced by capsaicin or its analogs.
Figure 4—figure supplement 3
Activation of TRPV1 I574A mutant by capsaicin and its analogs.
Representative whole-cell patch-clamp recordings of TRPV1 I574A mutant with Cap (top left), Cap-1 (top right), and Cap+1 (bottom left), and the concentration response curves. n = 4–6 cells.
Figure 5 with 6 supplements
Capsaicin analogs elicit a structurally similar permissive state of the TRPV1 ligand-binding pocket.
(A and B) Comparisons of the ligand-binding pocket permissive conformations induced by CAP-1 (A) or CAP+1 (B) to the cryo-EM structure of capsaicin-bound state (3J5R). The backbone RMSD of the top …
Figure 5—figure supplement 1
Pore radii of Cap-1 (A) and Cap+1 (B) models (left) with its distribution plot (right).
Red lines represent the top 30 models from docking simulations from each analog. Black line represents the pore radii distribution of 3J5R. SF, selectivity filter. A.U., arbitrary units.
Figure 5—figure supplement 2
TRPV1 currents induced by Cap and its analogs exhibit similar ion permeation properties.
(A) Summary table of Cap and its analogs. Px/Na+ - permeability ratio of cation x relative to 140 mM Na+. Erev – reversal potential. Permeability ratios were calculated using the …
Figure 5—figure supplement 3
Electrostatic potential distribution of TRPV1 in the capsaicin-bound state, with top and bottom views.
The ion permeation pathway can also be viewed from a side view of the vanilloid-binding pocket with a capsaicin molecule in pink. Blue represents more electropositive regions with red representing …
Figure 5—figure supplement 4
Electrostatic potential of TRPV1 ligand-binding pocket.
Side view of the electrostatic potential of TRPV1 (PDB: 5IRZ) with a lipid bound (top left) and a zoomed in view of the ligand-binding pocket (bottom left). Side view of the electrostatic potential …
Figure 5—figure supplement 5
Electrostatic potential of capsaicin and its analogs.
Dashed line marks the position of the carbonyl oxygen on the neck of the compounds. Scale bar represents the electrostatic potentials with blue being electropositive and red being electronegative.
Figure 5—figure supplement 6
Electrostatic potential maps of capsaicin and halogenated capsaicin analogs.
Electrostatic potential maps of capsaicin and halogenated capsaicin analogs (Br, Cl, I – at either five or six position) were generated using MultiWFN. Red represents more electronegative regions …
Author response image 1
Comparisons between 3J5R and 5IRX.
(Left) Per-residue structural deviation is plotted for the RMSD in Å between these two structures in the ligand-binding pocket. (Right) Structural overlay of 3J5R (cyan) and 5IRX (brown) between S1 …
Tables
Table 1
Comparison of ligand activation of wildtype and mutant mTRPV1 channels by capaicin analogs.
| Cap | Cap-1 | Cap+1 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| EC50 (µM) | K | N | EC50 (µM) | K | N | EC50 (µM) | K | N | ||
| WT | 0.14 ± 0.01 | 1.82 ± 0.22 | 4 | 0.10 ± 0.01 | 1.20 ± 0.05 | 3 | 4.31 ± 0.23* | 1.44 ± 0.11 | 4 | |
| T551V | 1.56 ± 0.20† | 1.74 ± 0.13 | 5 | 9.13 ± 1.10*‡ | 1.83 ± 0.31 | 5 | 70.12 ± 7.74*§ | 1.71 ± 0.10 | 8 | |
| E571A | 1.53 ± 0.10† | 1.86 ± 0.07 | 4 | 19.31 ± 0.22*‡ | 2.01 ± 0.02 | 6 | 31.43 ± 0.02*§ | 1.84 ± 0.11 | 6 | |
| I574A | 0.22 ± 0.02† | 1.41 ± 0.12 | 3 | 2.80 ± 0.11*‡ | 1.35 ± 0.23 | 7 | 10.73 ± 0.33*§ | 1.98 ± 0.12 | 5 | |
| T671S | 0.05 ± 0.06 | 0.87 ± 0.11 | 3 | 22.80 ± 0.40*‡ | 4.94 ± 0.30 | 3 | 35.29 ± 1.19*§ | 3.16 ± 0.45 | 3 | |
| Y512F | 1.12 ± 0.17† | 1.35 ± 0.08 | 4 | 25.18 ± 1.18*‡ | 2.68 ± 0.34 | 3 | 99.48 ± 6.82*§ | 1.46 ± 0.17 | 3 | |
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* Compared to Capsaicin (Cap).
†Compared to WT Cap.
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‡Compared to WT Cap-1.
§Compared to WT Cap+1.
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EC50, half maximal effective concentration; K, Hill coefficient; N, number of patches.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK 293 | ATCC | Cat #: CRL-1573 | |
| Chemical compound, drug | Capsaicin | Sigma | Cat #: M2028 | |
| Chemical compound | (E)−8-methylnon-6-enoyl chloride | TCI America | Cat #: 95636-02-5 Product: M1826 | |
| Chemical compound | 4-(2-aminoethyl)−2-methoxyphenol | TCI America | Cat #: 7149-10-2 Product: A2330 | |
| Chemical compound | 4-amino-2-methoxyphenol | TCI America | Cat #: 52200-90-5 Product: A2883 | |
| Software, algorithm | IgorPro | IgorPro (https://www.wavemetrics.com/) | Version 8 | |
| Software, algorithm | Rosetta | Rosetta (https://www.rosettacommons.org/) | Version 3.10 | |
| Software, algorithm | Chimera | UCSF Chimera (https://www.cgl.ucsf.edu/chimera/) | Version 1.14 | |
| Software, algorithm | VMD | VMD (https://www.ks.uiuc.edu/Research/vmd/) | Version 1.9.3 | |
| Software, algorithm | GAMESS | GAMESS (https://www.msg.chem.iastate.edu/GAMESS/) | Version Sept. 30,2018 R3 | |
| Software, algorithm | MultiWFN | MultiWFN (http://sobereva.com/multiwfn/) | Version 3.6 | |
| Software, algorithm | Avogadro | Avogadro (https://avogadro.cc/) | Version 1.2.0 | |
| Software, algorithm | OpenEye OMEGA | OpenEye OMEGA (https://www.eyesopen.com/omega) | Version 2.4.3 | |
| Software, algorithm | QuB | QuB (https://www.qub.buffalo.edu/) | Version 2.0.0.30 |
Additional files
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Supplementary file 1
Description of synthesis, 1H NMR, and 13C NMR of capsaicin analogs.
Rosetta commands and scripts for docking.
- https://cdn.elifesciences.org/articles/62039/elife-62039-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62039/elife-62039-transrepform-v2.pdf
