Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.
TRPV1; TRPV2; capsaicin; ligand gating; resiniferatoxin.
Conflict of interest statement
The authors declare no conflict of interest.
Rational design of point mutations in TRPV2 to introduce vanilloid sensitivity. (
A) Cryo-EM structure of TRPV1 (model 3J5R in PDB; electron density map, 5777 in EMD) shows that capsaicin (electron density colored in orange) binds to the transmembrane domains. Lipid membrane boundaries are indicated by cyan disks. ( B) Binding configuration of capsaicin. Two residues (in orange), T551 on S4 and E571 on the S4–S5 linker, form a hydrogen bond with the neck and head of capsaicin, respectively. Another two key residues, S513 and F544, are shown in yellow. ( C) Comparison of chemical structures of capsaicin and resiniferatoxin. The vanillyl head and tail groups are shades in yellow and green, respectively. The =O and –OH groups predicted to form a hydrogen bond with TRPV1 are shown in red. ( D) Sequence logo of the alignment between TRPV1 and TRPV2 channels. The height of a letter is proportional to the relative frequency of that residue at a particular site. Polar residues are colored in green, hydrophobic residues in black, positively charged residues in blue, and negatively charged residues in red. Four key residues designated for mutagenesis are marked by an arrow, with the corresponding residues in TRPV1 and TRPV2 shown in red and blue, respectively.
Multiple sequence alignment of TRPV1 and TRPV2 in different species. Only the transmembrane domains of these channels are shown. Alignment is performed by Clustal Omega and visualized in Jalview with Zappo color scheme. Four residues chosen for mutagenesis are marked by an arrow, with the residues in TRPV1 given in red.
Sequence Logo of the multiple sequence alignment. The alignment in Fig. S1 is analyzed by the Web Logo serve to calculate the relative frequency of amino acids at each site within the transmembrane domain. Polar residues are colored in green, positively charged residues in blue, negatively charged residues in red, and hydrophobic ones in black. Four residues chosen for mutagenesis are marked by an arrow, with the residues in TRPV1 given in red.
TRPV2_Quad is sensitive to resiniferatoxin. (
A) The capsaicin-binding pocket of TRPV1 (gray; PDB ID code 3J5R) is structurally aligned with that of TRPV2 (blue; PDB ID code 5AN8), with the side chain of the four key residues shown in red and orange, respectively. ( B) An example whole-cell recording demonstrates that TRPV2_Quad was not activated by capsaicin up to 10 µM, whereas 2-APB activated the channels in the same membrane patch. ( C) Resiniferatoxin activates TRPV2_Quad in a concentration-dependent manner in an inside-out patch. ( D) Averaged concentration dependence of resiniferatoxin activation ( n = 9) is fitted to a Hill equation with the following parameters: EC 50, 1.4 ± 0.2 µM; Hill coefficient, 1.5 ± 0.1. The resiniferatoxin responses are normalized to the 2-APB response of the same membrane patch. ( E) Capsaicin antagonizes resiniferatoxin activation of TRPV2_Quad in a concentration-dependent manner in an inside-out patch. ( F) Averaged concentration dependence of capsaicin inhibition ( n = 5) is fitted to a Hill equation with the following parameters: IC 50, 146.7 ± 15.2 nM; Hill coefficient, 0.7 ± 0.1. Note that, with increasing concentration of capsaicin, resiniferatoxin activation takes a longer time to reach equilibrium, which may lead to underestimation of the value of IC 50.
Resiniferatoxin activates TRPV1 but not TRPV2. (
A) Representative inside-out patch recording of TRPV1 activated by resiniferatoxin at both +80 mV ( Top) and −80 mV ( Bottom). Note that, although resiniferatoxin activation is effectively blocked by Ba 2+, it cannot be washed off by perfusion of bath solution. ( B) In this representative whole-cell recording, although TRPV2 channels were activated by 2-APB, these channels cannot be activated by resiniferatoxin up to 10 µM. ( C) The ratio of current amplitudes induced by 10 µM resiniferatoxin and 3 mM 2-APB between wild-type TRPV2 (0.02 ± 0.004; n = 4) and TRPV2_Quad (0.26 ± 0.03; n = 5) is significantly different (*** P < 0.001). The small current response to 10 µM resiniferatoxin in wild-type TRPV2 is largely due to nonspecific perturbation of membrane by the lipophilic resiniferatoxin.
Capsaicin does not inhibit 2-APB–induced activation of wild-type TRPV2 or TRPV3 but facilitates 2-APB–induced TRPV2_Quad activation. (
A) Representative current trace from an inside-out recording of wild-type TRPV2 activated by 2-APB in the absence and presence of capsaicin up to 30 µM at +80 mV. As time lapsed, there was a small decrease in current amplitude; however, when the current stabilized, application of 2-APB with or without capsaicin exhibited similar current amplitudes. The current ratio between last 2-APB application and 2-APB application with 10 µM capsaicin was used for statistical analysis shown in D. The initial decline in current amplitude is likely due to channel rundown or desensitization. It is, however, also possible that capsaicin may bind nonspecifically to the hydrophobic pocket, as the cryo-EM data of TRPV1 and TRPV2 indeed showed the presence of likely a lipid molecule at this location. ( B) Representative current trace from an inside-out recording of wild-type TRPV3 activated by 2-APB in the absence and presence of 10 µM capsaicin. No current inhibition by capsaicin was observed. ( C) Representative current trace from an inside-out recording of TRPV2_Quad activated by 2-APB in the absence and presence of 10 µM capsaicin. Capsaicin potentiated the 2-APB response, as capsaicin is able to bind to TRPV2_Quad. The 2-APB binding site in TRP channels is unknown to date. The potentiation is likely due to allosteric effects of capsaicin binding. ( D) Effects of capsaicin on resiniferatoxin and 2-APB–induced channel activation. On TRPV2_Quad, capsaicin inhibits resiniferatoxin activation as the current ratio is significantly smaller than 1 ( n = 5; *** P < 0.001) (Fig. 2 E and F), whereas it potentiates 2-APB response ( n = 6; ** P < 0.01). There is no significant effect of capsaicin on 2-APB activation of wild-type TRPV2 and TRPV3 ( n = 3 for each; N.S., no significance.)
Resiniferatoxin-induced TRPV2_Quad activation exhibits a large OFF response. (
A) TRPV2_Quad pretreated with 2-APB yielded a large transient current surge (marked with a red dashed box) upon removal of resiniferatoxin. Representative current traces at labeled time points are shown in Inset. ( B) A three-state gating model is sufficient to recapitulate the time course of the resiniferatoxin-induced TRPV2_Quad response. A double-exponential function dictated by such a gating model with two similar time constants such as 1.1 and 0.9 s was able to recapitulate the OFF response (dashed red trace). ( C) Based on the three-state gating model shown in B, the concentration dependence of steady-state open probability (Po) before wash-off is fitted to determine K and L, the resiniferatoxin binding affinity, and the equilibrium constant for the O←→ I transition, respectively (see Materials and Methods for details). K = 203.7 ± 36.1 nM −1; L = 4.9 ± 1.3. ( D) Averaged concentration dependence of the peak OFF response (red symbols; n = 6) is fitted to a Hill equation with an EC 50 value of 222.9 ± 44.3 nM ( n = 6). The concentration–response curve without pretreatment of 2-APB (Fig. 2 D) is reproduced here as the black dashed curve. Open probability is calculated by normalizing resiniferatoxin responses to the 2-APB response of the same membrane patch as the open probability achieved by 2-APB was determined by noise analysis (Fig. S7).
Resiniferatoxin induces no steady-state activation of TRPV2_Triple (
A) or TRPV2_Double mutant ( B), which lacks either mutation Q525E or two mutations Q525E and S498F in comparison with TRPV2_Quad. These two channels exhibited an OFF response when resiniferatoxin was washed off, but the current amplitude appeared much smaller than that of TRPV2_Quad. All currents were recorded at both +80 and −80 mV.
Single-channel conductance levels are similar when TRPV2_Quad is activated by either resiniferatoxin or 2-APB. (
A) Representative single-channel recordings and corresponding all-point histogram of TRPV2_Quad activated by resiniferatoxin ( Top) or 2-APB ( Bottom). ( B) Resiniferatoxin and 2-APB activate TRPV2_Quad to a similar conductance level (120.9 ± 1.4 and 119.9 ± 3.7 pS, respectively; n = 4 each). Single-channel currents were recorded at −80 mV.
Representative noise analysis of TRPV2_Quad current activated by 2-APB at 4 mM (saturating concentration) (
I max_2-APB). The maximum open probability is estimated to be 83.7 ± 2.7% ( n = 6).
Similar physical properties observed in the ligand-binding pocket of TRPV1, TRPV2, and TRPV2_Quad. All structures are aligned to the conformation of the capsaicin-binding pocket in TRPV1 (
Top Left), in which a capsaicin molecule (in red) is shown. Electrostatic potential is calculated by Adaptive Poisson–Boltzmann Solver (APBS) in UCSF Chimera. Positive and negative charged residues are colored in blue and red, respectively. Hydrophobicity is calculated based on the Kyte and Doolittle scale in UCSF Chimera. Hydrophobic and hydrophilic residues are colored in orange and blue, respectively. Two (S498F and Q525E) of the four mutations in TRPV2_Quad, as well as their corresponding wild-type residues, are marked by an arrow.
Structural difference in the binding pockets of TRPV1 and TRPV2. (
A) Front view of the ligand-binding pocket, with TRPV1 and TRPV2 shown in gray and blue, respectively. With their structures aligned by the S1–S4 domains, the S4–S5 linker of TRPV2 is about 3 Å lower than that of TRPV1. This leads to a 2.4-Å difference in distance between T551 and E571 in TRPV1 (colored in red; 11.5 Å) compared with that of the corresponding residues in TRPV2 (colored in orange; 13.9 Å). ( B) Side view of the pocket. The lowering of S4–S5 linker in TRPV2 is accompanied with an extra helical turn at the end of S4 (indicated by a red dashed box).
Docking of vanilloid molecules reveals a common mechanism for ligand activation. For each docking experiment, the model with the lowest binding energy among the converged cluster of top 10 models is shown. (
A) Docking of resiniferatoxin to TRPV1. The cryo-EM structure of TRPV1 with RTX bound (PDB ID code 3J5Q) was used. The tail of docked resiniferatoxin agreeably overlaps with the experimentally observed electron density (surface colored in yellow; EMD ID 5776). Y512 marks the entrance of the binding pocket. Four critical residues for capsaicin activation are in red. Potential hydrogen bonds between resiniferatoxin and TRPV1 are represented by black dashed lines. ( B) Docking of capsaicin to TRPV1. ( C) Docking of resiniferatoxin to TRPV2_Quad. Residues potentially forming hydrogen bonds with resiniferatoxin are in red. Note that resiniferatoxin is able to preserve a similar binding configuration as in TRPV1, with the tail residing inside the binding pocket and the head forming a network of hydrogen bonds. ( D) Docking of resiniferatoxin to wild-type TRPV2. Unlike in TRPV2_Quad ( C), resiniferatoxin cannot stay inside the binding pocket, as its tail is outside of Y466, which corresponds to Y512 in TRPV1 that marks the entrance of binding pocket. ( E) Docking of capsaicin to TRPV2_Quad. Compared with docking in TRPV1 ( B), the neck of capsaicin still forms a hydrogen bond with T505 (equivalent to T551 on TRPV1). Instead of E525 in the S4–S5 linker, multiple potential hydrogen bonds are observed between the head and Y466, S467, and R512. ( F) A cartoon illustrating that a vanilloid ligand, when bound favorably inside the binding pocket, stabilizes the S4–S5 linker toward S4, leading to the repositioning of S6 to open the TRP channel.
Converged configurations of docked vanilloid molecules. The largest cluster among the top 10 models with lowest binding energy is shown for each docking experiment. The model with lowest binding energy in the converged cluster is chosen as the final representative model shown in Fig. 6.
All figures (14)
Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):E137-45. doi: 10.1073/pnas.1517288113. Epub 2015 Dec 30.
Proc Natl Acad Sci U S A. 2016.
26719417 Free PMC article.
Understand spiciness: mechanism of TRPV1 channel activation by capsaicin.
Protein Cell. 2017 Mar;8(3):169-177. doi: 10.1007/s13238-016-0353-7. Epub 2017 Jan 2.
Protein Cell. 2017.
28044278 Free PMC article.
Engineering vanilloid-sensitivity into the rat TRPV2 channel.
Elife. 2016 May 13;5:e16409. doi: 10.7554/eLife.16409.
27177419 Free PMC article.
Differential effects of TRPV channel block on polymodal activation of rat cutaneous nociceptors in vitro.
Exp Brain Res. 2009 Jun;196(1):31-44. doi: 10.1007/s00221-009-1808-3. Epub 2009 Apr 30.
Exp Brain Res. 2009.
Structural mechanisms of transient receptor potential ion channels.
J Gen Physiol. 2020 Mar 2;152(3):e201811998. doi: 10.1085/jgp.201811998.
J Gen Physiol. 2020.
Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2.
Structure. 2020 Feb 4;28(2):169-184.e5. doi: 10.1016/j.str.2019.11.005. Epub 2019 Dec 2.
31806353 Free PMC article.
The ion selectivity filter is not an activation gate in TRPV1-3 channels.
Elife. 2019 Nov 14;8:e51212. doi: 10.7554/eLife.51212.
31724952 Free PMC article.
What is new about mild temperature sensing? A review of recent findings.
Temperature (Austin). 2019 Apr 29;6(2):132-141. doi: 10.1080/23328940.2019.1607490. eCollection 2019.
Temperature (Austin). 2019.
31286024 Free PMC article.
Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers.
Br J Pharmacol. 2019 Sep;176(17):3364-3377. doi: 10.1111/bph.14766. Epub 2019 Jul 22.
Br J Pharmacol. 2019.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Molecular Docking Simulation
TRPV Cation Channels / chemistry
TRPV Cation Channels / genetics
TRPV Cation Channels / metabolism*
LinkOut - more resources
Full Text Sources Other Literature Sources Molecular Biology Databases Miscellaneous