Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin

Elokely, Khaled M.; Velisetty, Phanindra; Delemotte, Lucie; Palovcak, Eugene; Klein, Michael L.; Rohács, Tibor; Carnevale, Vincenzo

doi:10.1073/pnas.1517288113

Cited by 138 publications

(161 citation statements)

References 52 publications

(56 reference statements)

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“…This is followed by the formation of another hydrogen bond between the head and E571, which stabilizes the outward conformation of S4-S5 linker to open the channel. Although this framework of ligand-gating mechanism is widely supported by the latest computational studies (13)(14)(15), further functional tests are needed.…”

mentioning

confidence: 99%

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Yang

Yarov‐Yarovoy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 Rosettabased 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.on channels constitute the second largest family of drug targets for therapeutics (1-3); therefore, understanding their gating mechanisms by small-molecule ligands is critical for both basic and translational research. Capsaicin activation of the pain-sensing transient receptor potential vanilloid 1 (TRPV1) ion channel represents an outstanding model system for understanding liganddependent gating process (4), because capsaicin not only potently activates the channel with a submicromolar EC 50 (5) but also effectively stabilizes the channel at high open probability (6-8). Moreover, capsaicin activation is highly selective for TRPV1 channel (5). Previous mutagenesis (9, 10) and cryo-EM studies (11,12) have shown that capsaicin binds near the third (S3) and fourth (S4) transmembrane segments of TRPV1 (Fig. 1A). Based on the high-resolution cryo-EM structures, we have used a combination of computational and functional assays to reveal that capsaicin takes a "tail-up, head-down" configuration in the ligand-binding pocket (6) (Fig. 1B). The aliphatic tail forms extensive but nonspecific van der Waals (VDW) interactions with residues lining the binding pocket, whereas the vanillyl group (head) and the amide group (neck) of capsaicin form a hydrogen bond with E571 on S4-S5 linker and T551 on S4, respectively. To activate TRPV1, VDW interactions first secure capsaicin in the pocket and the neck of capsaicin forms a hydrogen bond with T551. This is followed by the formation of another hydrogen bond between the head and E571, which stabilizes the outward conformation of S4-S5 linker to open the chann...

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mentioning

confidence: 99%

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Yang

Yarov‐Yarovoy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The S4-S5 linker forms a horizontal helix connecting the peripheral S1-S4 domain to the central pore domain 22 . Recent studies confirmed that it serves a critical role in coupling capsaicin binding to channel activation [16][17][18][19] . We made a series of insertions of 2-to-12 amino acids in length into this key structure, at three different positions (Fig.…”

Section: Functional Tests Of Insertion Mutantsmentioning

confidence: 88%

“…Among TRPV1 stimulations, the capsaicin-channel interaction is the best-understood case in both location and detailed atomic interactions [14][15][16][17][18][19] . None of the functional insertion mutations disrupted capsaicin activation, in agreement with the notion that capsaicin-activation machinery mostly resides within the transmembrane region.…”

Section: Discussionmentioning

confidence: 99%

Exploring Functional Roles of TRPV1 Intracellular Domains with Unstructured Peptide-Insertion Screening

Yang

et al. 2017

Biophysical Journal

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TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli that interact with different parts of the channel protein. Recent cryo-EM studies revealed detailed channel structures, opening the door for mapping structural elements mediating activation by each stimulus. Towards this goal, here we have combined unstructured peptide-insertion screening (UPS) with electrophysiological and fluorescence recordings to explore structural and functional roles of the intracellular regions of TRPV1 in mediating various activation stimuli. We found that most of the tightly packed protein regions did not tolerate structural perturbation by UPS when tested, indicating that structural integrity of the intracellular region is critical. In agreement with previous reports, Ca 2+ -dependent desensitization is strongly dependent on both intracellular N-and C-terminal domains; insertions of an unstructured peptide between these domains and the transmembrane core domain nearly eliminated Ca 2+ -dependent desensitization. In contrast, channel activations by capsaicin, low pH, divalent cations, and even heat are mostly intact in mutant channels containing the same insertions. These observations suggest that the transmembrane core domain of TRPV1, but not the intracellular domains, is responsible for sensing these stimuli.TRPV1 ion channel can be directly activated or modulated by capsaicin 1 , heat 1 , extracellular proton 2 , divalent cations 3 , Na + 4 , peptide toxins from spider 5 , centipede 6 , and scorpion 7 , membrane depolarization 8 , intracellular Ca 2+ 9 , and many other factors 10 . A noticeable feature of TRPV1 polymodal activation emerging from biophysical investigations is the existence of non-overlapping activation pathways [11][12][13] . However, except for capsaicin 14-19 , proton 11,20 , and animal toxins 6,21 , the channel structures that sense activation stimuli are poorly defined. Recent cryo-EM studies revealed that the transmembrane core domain of TRPV1 resembles that of tetrameric cation channels, with six transmembrane helical segments surrounding a centrally located ion permeation pore 22,23 . The partially resolved intracellular N-and C-terminal domains contain special structures such as the ankyrin-like repeat domains and the TRP domain that likely play crucial structural and/or functional roles 10 . Arrangement of intracellular domains has been investigated using fluorescence resonance energy transfer 24 . A major task for TRPV1 study in the post-structure era is to assign functional roles to individual structure domains.The unstructured peptide-insertion screening (UPS) strategy had been used to rapidly and reliably obtain information on structure-function relationships in an ion channel even before detailed protein structures were available. In one effective application, isolating the pore domain of yeast TRPY1 channel from the intracellular Ca 2+ -sensing domain with unstructured peptides demonstrated that the pore domain was mechanosensitive 25 . Similarly, unstructured peptide insertion...

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“…Numerous studies have indicated that glutamic acid is involved in binding of the vanillyl ring of vanilloid structures to the vanilloid receptor, TRPV1, interacting specifically with the hydroxyl functional group. [27][28][29] Thus, acetic acid may demonstrate a possible methods of interaction between the vanillyl ring and cellular molecules. In addition, the protonated form of acetic acid was used rather than the ionized form, in order to stabilize the molecule and allow it to interact with the phenol derivatives without the use of an explicit water model.…”

Section: Rationale Behind Chosen Structuresmentioning

confidence: 99%

Ortho-Methoxy Group as a Mild Inhibitor of the Reactions Between Carboxylic Acid and Phenols

et al. 2017

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According to the current database of natural products, over 25,000 compounds contain a vanillyl ring in their structure. The reasoning behind the high occurrence of the vanillyl ring structure seemed to be poorly understood, specifically the preference for a methoxy-substituted phenol structure as opposed to its dihydroxy analogue. To better understand this, we investigated the reaction mechanisms of two methoxyphenol structures, in syn and anti conformations, two hydroxyphenol structures, also in syn and anti conformations, and phenol as a reference structure, with acetic acid. Of the starting structures, the syn hydroxyphenol was found to be kinetically the most reactive, and formed the most stable product, while both hydroxyl-substituted phenols reacted more favorably with acetic acid than the methoxyphenols. A preference for the methoxyphenol molecule may exist as a way to hinder the formation of stable covalent bonds between natural products and cellular components.

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Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin

Cited by 138 publications

References 52 publications

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Exploring Functional Roles of TRPV1 Intracellular Domains with Unstructured Peptide-Insertion Screening

Ortho-Methoxy Group as a Mild Inhibitor of the Reactions Between Carboxylic Acid and Phenols

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