If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* 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.
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.
). 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) (
). 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.
). 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 (
). 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 (
). 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 (
). 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) (
), 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.
Taking into account the suggested rotation of the rTRPV1 Tyr511 residue (
), 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.
Endogenous and exogenous vanilloids activate sensory nerve terminals by opening TRPV1 channels, eliciting receptor generator potentials, initiating action potentials, and leading to pain sensation (
). 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 (
), 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 (
). 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 (
)) (Equation 2), which separates ligand binding from activation gating of ligand-gated ion channels,
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”) (
). 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 (
), 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 (
) (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 (
), 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 (
). 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.
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.
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.
The vanilloid receptor: a molecular gateway to the pain pathway.