title: Tyrosine Residue in the TRPV1 Vanilloid Binding Pocket Regulates Deactivation Kinetics.
abstract: 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 (Tyr(511)) 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 Tyr(511) 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 Tyr(511) substitutions, and tested their response to both exo- and endovanilloids. Our data show that only substitutions of Tyr(511) 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 Tyr(511) 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 Tyr(511) and the agonist. To conclude, we suggest that Tyr(511)-mediated anchoring of vanilloids in their binding pocket is pivotal for TRPV1 activation and subsequent pain sensation.
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Kumar
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Adina
authors.initials: A
authors.lastname: Hazan
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Arijit
authors.initials: A
authors.lastname: Basu
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Nomi
authors.initials: N
authors.lastname: Zalcman
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors.forename: Henry
authors.initials: H
authors.lastname: Matzner
authors.affiliation: From the Faculty of Medicine, Institute for Drug Research (IDR), School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel avip@ekmd.huji.ac.il.
authors.forename: Avi
authors.initials: A
authors.lastname: Priel
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D010146:Pain / Q000235:genetics / Q000378:metabolism; D051381:Rats; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine
chemical_list: D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C493922:Trpv1 protein, rat; D014443:Tyrosine
keywords: electrophysiology; gating; site-directed mutagenesis; structure-function; transient receptor potential channels (TRP channels)