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Tyrosine Residue in the TRPV1 Vanilloid Binding Pocket Regulates Deactivation Kinetics*

  • Rakesh
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Adina Hazan
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Arijit Basu
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Nomi Zalcman
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Henry Matzner
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Avi Priel
    Correspondence
    To whom correspondence should be addressed. Tel.: 972-2-6757299; Fax: 972-2-6757339
    Affiliations
    From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
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  • Author Footnotes
    * This work was supported in part by the Israel Science Foundation Grants 1721/12 and 1368/12 (to A. P.), Marie-Curie Career Integration Grant FP7-CIG-321899 (to A. P.), a Jerusalem Brain Committee (JBC) Postdoctoral Fellowship (to R. K.), the Kaete Klausner fellowship (to A. H), and the Fellowship Program for Outstanding Post-Doctoral Researchers of the Planning and Budget Commission of Israel (to A. B.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:May 03, 2016DOI:https://doi.org/10.1074/jbc.M116.726372
      Vanilloids are pain evoking molecules that serve as ligands of the “heat and capsaicin receptor” TRPV1. Binding of either endogenous or exogenous vanilloids evokes channel and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation and deactivation is not fully understood. The highly conserved tyrosine in position 511 (Tyr511) of the rat TRPV1 (rTRPV1) was the first residue to be identified as a necessary participant in the vanilloid-mediated response. rTRPV1 cryo-EM structures implicated rotation of this residue in the vanilloids bound state. Therefore, we hypothesize that the rTRPV1 Tyr511 residue entraps vanilloids in their binding site, prolonging channel activity. To test our hypothesis, we generated an array of rTRPV1 mutants, containing the whole spectrum of Tyr511 substitutions, and tested their response to both exo- and endovanilloids. Our data show that only substitutions of Tyr511 to aromatic amino acids were able to mimic, albeit partially, the vanilloid-evoked activation pattern of the wt receptor. Although these substitutions reduced the channel sensitivity to vanilloids, a maximal open-channel lifetime could be achieved. Moreover, whereas their current activation rate remains intact, receptors with Tyr511 substitutions exhibited a faster current deactivation. Our findings therefore suggest that the duration of channel activity evoked by vanilloids is regulated by the interaction between Tyr511 and the agonist. To conclude, we suggest that Tyr511-mediated anchoring of vanilloids in their binding pocket is pivotal for TRPV1 activation and subsequent pain sensation.

      Introduction

      Vanilloids, both endogenous and exogenous, are pain evoking molecules (
      • Caterina M.J.
      • Julius D.
      The vanilloid receptor: a molecular gateway to the pain pathway.
      ,
      • Van Der Stelt M.
      • Di Marzo V.
      Endovanilloids: putative endogenous ligands of transient receptor potential vanilloid 1 channels.
      ). They serve as ligands of the mammalian transient receptor potential vanilloid type 1 (TRPV1)
      The abbreviations used are: rTRPV1
      rat transient receptor potential vanilloid 1
      2-APB
      2-aminoethoxydiphenyl borate
      Cap
      capsaicin
      NADA
      N-arachidonoyl dopamine
      VBS
      vanilloid binding site
      PDB
      Protein Data Bank
      ANOVA
      analysis of variance.
      protein, a nonselective cation channel also known as the “heat and capsaicin receptor” (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      The capsaicin receptor: a heat-activated ion channel in the pain pathway.
      ). TRPV1 is mainly expressed on C and Aδ fibers of the somatosensory system, where it plays an essential role in the development of inflammatory hyperalgesia and pain (
      • Basbaum A.I.
      • Bautista D.M.
      • Scherrer G.
      • Julius D.
      Cellular and molecular mechanisms of pain.
      ,
      • Dubin A.E.
      • Patapoutian A.
      Nociceptors: the sensors of the pain pathway.
      ,
      • Julius D.
      TRP channels and pain.
      ). Furthermore, this polymodal receptor acts as a molecular sensor for a large array of acute noxious stimuli, of both physical and chemical nature, including, in addition to vanilloids, heat (>42 °C) (
      • Caterina M.J.
      Transient receptor potential ion channels as participants in thermosensation and thermoregulation.
      ), low pH (pH ≤ 6.5) (
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilbert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      The cloned capsaicin receptor integrates multiple pain-producing stimuli.
      ,
      • Ryu S.
      • Liu B.
      • Qin F.
      Low pH potentiates both capsaicin binding and channel gating of VR1 receptors.
      ), and peptide toxins (
      • Siemens J.
      • Zhou S.
      • Piskorowski R.
      • Nikai T.
      • Lumpkin E.A.
      • Basbaum A.I.
      • King D.
      • Julius D.
      Spider toxins activate the capsaicin receptor to produce inflammatory pain.
      ,
      • Bohlen C.J.
      • Priel A.
      • Zhou S.
      • King D.
      • Siemens J.
      • Julius D.
      A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain.
      ). Although endovanilloids (such as the endocannabinoids anandamide, N-arachidonoyl dopamine (NADA), and lipoxygenase products of arachidonic acid) have been identified as TRPV1 agonists (
      • Zygmunt P.M.
      • Petersson J.
      • Andersson D.A.
      • Chuang H.
      • Sørgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.D.
      • Marzo V. Di
      • Ho E.D.
      Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide.
      ,
      • Piomelli D.
      • Sasso O.
      Peripheral gating of pain signals by endogenous lipid mediators.
      ), its most known and studied activator is the exovanilloid capsaicin, the “hot” ingredient in chili peppers (
      • O'Neill J.
      • Brock C.
      • Olesen A.E.
      • Andresen T.
      • Nilsson M.
      • Dickenson A.H.
      Unravelling the mystery of capsaicin: a tool to understand and treat pain.
      ). However, although elucidating this ligand-receptor interaction will provide a better understanding of the mechanism underlying noxious stimuli detection in the pain pathway, the molecular basis of vanilloids-mediated TRPV1 activation and deactivation is not fully understood.
      Since TRPV1 cloning in 1997 (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      The capsaicin receptor: a heat-activated ion channel in the pain pathway.
      ), several residues that participate in its vanilloid-mediated activation have been identified (
      • Jordt S.E.
      • Julius D.
      Molecular basis for species-specific sensitivity to “hot” chili peppers.
      ,
      • Chou M.Z.
      • Mtui T.
      • Gao Y.-D.
      • Kohler M.
      • Middleton R.E.
      Resiniferatoxin binds to the capsaicin receptor (TRPV1) near the extracellular side of the S4 transmembrane domain.
      ,
      • Gavva N.R.
      • Klionsky L.
      • Qu Y.
      • Shi L.
      • Tamir R.
      • Edenson S.
      • Zhang T.J.
      • Viswanadhan V.N.
      • Toth A.
      • Pearce L.V.
      • Vanderah T.W.
      • Porreca F.
      • Blumberg P.M.
      • Lile J.
      • Sun Y.
      • Wild K.
      • Louis J.-C.
      • Treanor J.J.
      Molecular determinants of vanilloid sensitivity in TRPV1.
      ,
      • Boukalova S.
      • Marsakova L.
      • Teisinger J.
      • Vlachova V.
      Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels.
      ,
      • Winter Z.
      • Buhala A.
      • Ötvös F.
      • Jósvay K.
      • Vizler C.
      • Dombi G.
      • Szakonyi G.
      • Oláh Z.
      Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel: an overview of the current mutational data.
      ). The identification of these residues, mainly through site-directed mutagenesis analyses, laid the basis for the ever increasing body of studies aiming to elucidate various aspects of this important receptor (
      • Szallasi A.
      • Cortright D.N.
      • Blum C.A.
      • Eid S.R.
      The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept.
      ,
      • Vriens J.
      • Appendino G.
      • Nilius B.
      Pharmacology of vanilloid transient receptor potential cation channels.
      ,
      • Bevan S.
      • Quallo T.
      • Andersson D.A.
      TRPV1.
      ). These studies have led to the identification of the vanilloid binding site (VBS), a TRPV1 intracellular pocket to which all known vanilloids, endogenous and exogenous, bind and evoke subsequent channel activation (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ,
      • Hanson S.M.
      • Newstead S.
      • Swartz K.J.
      • Sansom M.S.
      Capsaicin interaction with TRPV1 channels in a lipid bilayer: molecular dynamics simulation.
      ,
      • Hazan A.
      • Kumar R.
      • Matzner H.
      • Priel A.
      The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.
      ,
      • Yang F.
      • Xiao X.
      • Cheng W.
      • Yang W.
      • Yu P.
      • Song Z.
      • Yarov-Yarovoy V.
      • Zheng J.
      Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ). The solved cryo-EM structure of the rat TRPV1 (rTRPV1) showed that in the presence of vanilloids (capsaicin or resiniferatoxin), residues within the VBS are scattered around the agonist, in proximity to the intracellular domain between the receptor S3 and S4 transmembrane segments (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ). Moreover, based on this cryo-EM structure, a recent molecular docking and molecular dynamic studies proposed potential configurations of vanilloids inside the VBS (
      • Yang F.
      • Xiao X.
      • Cheng W.
      • Yang W.
      • Yu P.
      • Song Z.
      • Yarov-Yarovoy V.
      • Zheng J.
      Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ). However, the VBS mechanism of action and the roles of its different residues in the vanilloid-evoked TRPV1 response are yet to be defined.
      The tyrosine at position 511 of the rTRPV1 was the first VBS residue to be identified as a participant in capsaicin and resiniferatoxin-evoked receptor activation (
      • Jordt S.E.
      • Julius D.
      Molecular basis for species-specific sensitivity to “hot” chili peppers.
      ), as the Y511A substitution alone was sufficient to abolish receptor activation by these exogenous vanilloids (
      • Jordt S.E.
      • Julius D.
      Molecular basis for species-specific sensitivity to “hot” chili peppers.
      ,
      • Gavva N.R.
      • Klionsky L.
      • Qu Y.
      • Shi L.
      • Tamir R.
      • Edenson S.
      • Zhang T.J.
      • Viswanadhan V.N.
      • Toth A.
      • Pearce L.V.
      • Vanderah T.W.
      • Porreca F.
      • Blumberg P.M.
      • Lile J.
      • Sun Y.
      • Wild K.
      • Louis J.-C.
      • Treanor J.J.
      Molecular determinants of vanilloid sensitivity in TRPV1.
      ). The rTRPV1 cryo-EM structures in the presence of exovanilloids clearly points to a close proximity of this highly conserved residue to the agonist (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ). Moreover, these structures indicate that Tyr511 assumes two distinct rotamers in apo versus bound states, where its side chain points away either from or into the VBS, respectively (Fig. 1A). Although recent molecular docking and molecular dynamics studies point to an interaction between Tyr511 and the ligand in the bound state (Fig. 1B) (
      • Yang F.
      • Xiao X.
      • Cheng W.
      • Yang W.
      • Yu P.
      • Song Z.
      • Yarov-Yarovoy V.
      • Zheng J.
      Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ), its position in the apo state suggests a limited role in the initial interaction between vanilloids and their binding site. Therefore, how this residue participates in the vanilloids-evoked TRPV1 activation remains to be determined.
      Figure thumbnail gr1
      FIGURE 1Substitutions in position 511 of rTRPV1 differentially affect capsaicin-evoked response. A, ribbon presentations of top views of rTRPV1 putative VBS in the apo (left; PDB code 3j5p; gray) and capsaicin-bound (right; PDB code 3j5r; blue) states. Tyr511, Thr550, and Glu570 residues are shown as sticks. Note the dramatic rotamer shift of Tyr511. B, a representative configuration of capsaicin-docked VBS (Cap; green). Tyr511, Thr550, and Glu570 residues are shown as sticks. Hydrogen bonds are shown as dashed black lines. C, representative calcium imaging analysis of rTRPV1 response. Left: pseudo-colored images of Fura-2-loaded HEK293T cells expressing the wt rTRPV1 (top panel), and the mutant receptors rTRPV1 (Y511W) (middle panel) and rTRPV1 (Y511I) (bottom panels) before (basal) and after application of capsaicin (2 μm), and after application of 2-APB (0.3 mm). Scale bar indicates levels of intracellular calcium. Right: changes with time of intracellular calcium levels of HEK293T cells expressing the wt (black line), Y511W (red line), Y511I (orange line), and Y511G (blue line) receptors in response to 2 μm capsaicin (empty bar), followed by subsequent application of 0.3 mm 2-APB (gray bar). All graphs represent an average of 50 2-APB responsive cells. D, box and whiskers plot shows capsaicin (2 μm)-evoked calcium response of HEK293T cells expressing rTRPV1 with the indicated Tyr511 substitutions, normalized to the 2-APB (0.3 mm)-evoked response. Boxes represent the mean of 3–4 independent experiments (each n ≥ 50 cells). Statistical significance between normalized responses of wt rTRPV1 and different mutant constructs are indicted as ***, p ≤ 0.001 (ANOVA followed by multiple comparison test).
      Taking into account the suggested rotation of the rTRPV1 Tyr511 residue (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ) and its interaction with vanilloid in the bound state (
      • Yang F.
      • Xiao X.
      • Cheng W.
      • Yang W.
      • Yu P.
      • Song Z.
      • Yarov-Yarovoy V.
      • Zheng J.
      Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ), we hypothesize that this residue entraps the vanilloids in the VBS, thus prolonging ligand occupancy in its binding pocket and enabling full duration of channel activation. To test this hypothesis, we generated an array of rTRPV1 mutants, containing the whole spectrum of Tyr511 substitutions, and tested their response to the exovanilloid capsaicin and the endovanilloid NADA by calcium imaging and electrophysiology. Our data showed that only substitutions of Tyr511 to aromatic amino acids were able to mimic, albeit partially, the vanilloid-evoked activation pattern of the wt receptor. Although these substitutions reduced the channel sensitivity to vanilloids, indicated by the rightward shift in capsaicin dose-response, maximal open-channel lifetime was achieved. Moreover, whereas current activation rate remains intact, Tyr511 substitutions lead to a faster current deactivation. Taken together, our data clearly indicate that upon vanilloid binding, Tyr511 rotates and interacts with the ligand to enable a full duration of channel activity. Thus, Tyr511 primarily regulates the deactivation process of vanilloid-mediated TRPV1 response. Furthermore, our findings suggest that substituting this residue to small aliphatic amino acids (such in the widely used Y511A substitution) shorten the duration of ligand occupancy in the VBS, which does not allow adequate channel gating. To conclude, we propose that upon its rotation, Tyr511 binds vanilloids to stabilize ligand-receptor interaction and allow vanilloid-evoked TRPV1 activation and pain sensation.

      Discussion

      Endogenous and exogenous vanilloids activate sensory nerve terminals by opening TRPV1 channels, eliciting receptor generator potentials, initiating action potentials, and leading to pain sensation (
      • Van Der Stelt M.
      • Di Marzo V.
      Endovanilloids: putative endogenous ligands of transient receptor potential vanilloid 1 channels.
      ,
      • Julius D.
      TRP channels and pain.
      ,
      • Vriens J.
      • Appendino G.
      • Nilius B.
      Pharmacology of vanilloid transient receptor potential cation channels.
      ). Although efforts to elucidate the molecular basis of these sensory processes have identified several amino acids in the TRPV1 protein that participate in vanilloids binding and subsequent channel gating (
      • Jordt S.E.
      • Julius D.
      Molecular basis for species-specific sensitivity to “hot” chili peppers.
      ,
      • Gavva N.R.
      • Klionsky L.
      • Qu Y.
      • Shi L.
      • Tamir R.
      • Edenson S.
      • Zhang T.J.
      • Viswanadhan V.N.
      • Toth A.
      • Pearce L.V.
      • Vanderah T.W.
      • Porreca F.
      • Blumberg P.M.
      • Lile J.
      • Sun Y.
      • Wild K.
      • Louis J.-C.
      • Treanor J.J.
      Molecular determinants of vanilloid sensitivity in TRPV1.
      ,
      • Winter Z.
      • Buhala A.
      • Ötvös F.
      • Jósvay K.
      • Vizler C.
      • Dombi G.
      • Szakonyi G.
      • Oláh Z.
      Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel: an overview of the current mutational data.
      ), their mechanism(s) of action remained largely unknown. Here, we analyzed the role of a conserved tyrosine, Tyr511 in the rTRPV1 sequence, in the vanilloid-evoked response. We show that substitutions to other aromatic residues only partly mimic tyrosine activity, implying that this amino acid directly interacts with the agonist. Furthermore, we provide evidence that rTRPV1 Tyr511 secures the agonist in its binding pocket, allowing full duration of the vanilloid-evoked response.
      Tyr511 of rTRPV1 was the first amino acid to be identified as part of the VBS (
      • Jordt S.E.
      • Julius D.
      Molecular basis for species-specific sensitivity to “hot” chili peppers.
      ). Although the Y511A substitution was extensively used to study physiological, structural, and biophysical processes related to the VBS, the exact role of this residue in the activation mechanism of TRPV1 by vanilloids remained largely unknown. To outline the specific role of Tyr511 in the vanilloid-evoked TRPV1 response, we initially substituted this residue with all of the different amino acids. Our findings clearly indicate that only substitution to the aromatic residues, phenylalanine and tryptophan, can mimic, albeit partly, the vanilloid-evoked response of the wt receptor (Figs. 1, 2, and 6). Although receptors containing substitutions to other aliphatic residues such as isoleucine, methionine, and cysteine were also activated by capsaicin and NADA, they did so to a much lower extent than substitutions to aromatic residues, whereas no activation was obtained upon substitutions to small aliphatic, charged or polar amino acids (Figs. 1, 2, and 6).
      A possible caveat of our analysis is the limited solubility of vanilloids, which precludes analysis in response to high concentrations. Therefore, it is possible that mutated receptors that did not respond to 30 μm capsaicin in our analysis maintained low sensitivity to this agonist and are capable to respond to higher concentrations. However, all of the known VBS-associated activators or inhibitors are highly hydrophobic and the membrane proximity of this site indicates that more hydrophilic agents might not reach this site (
      • Van Der Stelt M.
      • Di Marzo V.
      Endovanilloids: putative endogenous ligands of transient receptor potential vanilloid 1 channels.
      ). Nevertheless, our findings demonstrate that a maximal vanilloid-evoked TRPV1 activation could only be achieved if an aromatic residue occupies position 511.
      What is the role of Tyr511 in the activation mechanism of TRPV1 by vanilloids? An elegant, simplified model was proposed by Del Castillo and Katz (
      • Del Castillo J.
      • Katz B.
      Interaction at end-plate receptors between different choline derivatives.
      ) (recently summarized by William Zagotta (
      • Zagotta W.N.
      Ligand-dependent gating mechanism.
      )) (Equation 2), which separates ligand binding from activation gating of ligand-gated ion channels,
      MathJax Formula fx2
      (Eq. 2)


      where A is the agonist, R is the receptor, KA is the association equilibrium constant for binding, and L is the equilibrium constant of the bound channel. The parallel shift in the apparent EC50 of receptors containing the Y511F or Y511W substitutions (with no change in the Hill coefficient; Table 1 and Fig. 3), together with their unaffected open probability (at maximal capsaicin concentration; Fig. 4), clearly indicate the binding (KA) was most affected in these TRPV1 mutants (“K phenotype”) (
      • Galzi J.L.
      • Edelstein S.J.
      • Changeux J.
      The multiple phenotypes of allosteric receptor mutants.
      ). Furthermore, we found a dramatic shift of ∼2–3-fold in the deactivation rate, but no shift in the activation rate under saturation conditions of both capsaicin and NADA (FIGURE 5, FIGURE 6). Thus, our results point to a pivotal role of rTRPV1 Tyr511 in ligand binding, mainly in stabilizing the ligand-receptor complex, with no apparent role in receptor gating.
      Our data suggest that Tyr511 entraps the agonist to its binding pocket. Thus, agonist binding leads to movement of this residue, as suggested by the recently published rTRPV1 structure and molecular docking and dynamic analyses (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ,
      • Yang F.
      • Xiao X.
      • Cheng W.
      • Yang W.
      • Yu P.
      • Song Z.
      • Yarov-Yarovoy V.
      • Zheng J.
      Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ), resulting in favorable interactions and stabilization of the agonist in its pocket. Surprisingly, substitutions of tyrosine in this position with either phenylalanine or tryptophan caused a comparable shift in the apparent EC50 (Fig. 3) and current washout (FIGURE 5, FIGURE 6). This may reflect a role of the tyrosine hydroxyl group in anchoring the agonist. Indeed, the formation of a hydrogen bond between the Tyr511 hydroxyl group and capsaicin amide oxygen was suggested by a recently reported molecular dynamics simulation (
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ) (Fig. 1B). Our docking analysis suggests that the aromatic interaction between the phenyl ring of capsaicin and rTRPV1 Tyr511 allows a significant gain in free energy (−6 kcal/mol, with an average distance of ∼2 Å), which was further synergized by the hydrogen bond (by −1 kcal/mol). However, whereas the docking score of the Y511F substitution (−4.1 kcal/mol) was only marginally lower as compared with the wt receptor (−4.8 kcal/mol), its free energy gain was significantly lower (−2 kcal/mol, with an average distance of ∼2.5 Å). These observations suggest that whereas aryl-aryl interactions with aromatic rings favor vanilloid binding, the hydrogen bond between the agonist and the tyrosine residue further strengthen this interaction.
      Thus, in combination with the structure analysis and molecular dynamic simulation (
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ,
      • Darré L.
      • Domene C.
      Binding of capsaicin to the TRPV1 ion channel.
      ,
      • Elokely K.
      • Velisetty P.
      • Delemotte L.
      • Palovcak E.
      • Klein M.L.
      • Rohacs T.
      • Carnevale V.
      Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin.
      ), our mutagenesis analysis points to a two-step mechanism of Tyr511-depdendent TRPV1 activation. First, agonist binding induces conformational changes, resulting in an aryl-aryl interaction between the ligand and Tyr511 and enabling channel activation (as evident by the similar activation pattern of the aromatic substitutions, FIGURE 4, FIGURE 5). Ligand-receptor interactions involving aromatic rings, as we propose for vanilloids and Tyr511 of TRPV1, are key processes in biological recognition (
      • Meyer E.A.
      • Castellano R.K.
      • Diederich F.
      Interaction with aromatic rings in chemical and biological recognition.
      ). Second, the formation of hydrogen bonds between Tyr511 and the agonist, which prolongs agonist occupancy in the VBS (as suggested by the increased deactivation rate upon substitution to other aromatic acids, FIGURE 5, FIGURE 6). In summary, we suggest that TRPV1 Tyr511 is required for anchoring the agonist to the VBS through both aryl-aryl interaction and hydrogen bond formation, which increase the duration of the ligand-receptor complex, enabling adequate channel gating.

      Author Contributions

      A. P. conceived, coordinated the study, and wrote the paper. R. K. designed, performed, and analyzed the experiments in FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5. A. H. designed, performed, and analyzed the experiments in Fig. 6. A. B. and N. Z. designed, performed, and analyzed the experiments in Fig. 1. H. M. provided technical assistance and contributed to the electrophysiological analysis. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Amiram Goldblum for help with the structural analysis and members of the Priel laboratory for helpful discussion and comments. A. P. is affiliated with the Brettler Center for Research in Molecular Pharmacology and Therapeutics and the David R. Bloom Center for Pharmacy, School of Pharmacy, The Hebrew University of Jerusalem.

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