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.Vanilloids, both endogenous and exogenous, are pain evoking molecules (1, 2). They serve as ligands of the mammalian transient receptor potential vanilloid type 1 (TRPV1) 2 protein, a nonselective cation channel also known as the "heat and capsaicin receptor" (3). TRPV1 is mainly expressed on C and A␦ fibers of the somatosensory system, where it plays an essential role in the development of inflammatory hyperalgesia and pain (4 -6). 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 (Ͼ42°C) (7), low pH (pH Յ 6.5) (8, 9), and peptide toxins (10, 11). Although endovanilloids (such as the endocannabinoids anandamide, N-arachidonoyl dopamine (NADA), and lipoxygenase products of arachidonic acid) have been identified as TRPV1 agonists (12, 13), its most known and studied activator is the exovanilloid capsaicin, the "hot" ingredient in chili peppers (14). However, although elucidating this ligandreceptor 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 (3), several residues that participate in its vanilloid-mediated activ...

Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers

“…Key atomic interactions include two hydrogen bonds—formed between the amide group in the capsaicin neck and the hydroxyl group of T551 on the S4 segment of the TRPV1 channel (using mTRPV1 amino acid number) and between the hydroxyl group in the capsaicin head and the carboxyl group of E571 on the S4‐S5 linker—as well as extensive van der Waals (VDW) interactions including those by the capsaicin tail. Accuracy of the current capsaicin binding model has been satisfactorily confirmed by studies of the structural correlates of deactivation kinetics (Kumar et al, ), the activation conformational wave in TRPV1 channels triggered by capsaicin binding (Yang et al, ), the evolutionary drive for the tree shrew's insensitivity to spiciness (Han et al, ), and the rational designs of vanilloid‐sensitive TRPV2 channel mutants (Yang, Vu, Yarov‐Yarovoy, & Zheng, ; Zhang et al, ).…”

Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation

“…Cell culture and transfection were carried out as described (29). In brief, HEK293T cells were transfected with a total of 1 mg DNA using Mirus LT1 transfection reagent (Mirus Bio, Madison, WI, USA) according to manufacturer protocol.…”

“…Voltage-clamp recordings from TG neurons and transfected HEK293T cells were performed as previously described (15,29). In brief, membrane currents were recorded under the voltage clamp by using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA).…”

“…Live-cell calcium imaging was done as previously described (15,29). In brief, transfected HEK293T cells were spotted at PDL (0.2 mg/ml)-coated imaging chambers (m-slide, 8-well; Ibidi, Martinsried, Germany) and incubated for 1.5-2 h in a humidified incubator at 37°C and 5% CO 2 .…”

“…This configuration causes minimal perturbation of the intracellular milieu (46), which enables the recording of the activity of a single TRPV1 channel downstream of the Gq/GPCR signaling cascade. To determine the number of TRPV1 channels in each recorded patch, we analyzed their capsaicin-evoked response at the end of each experiment, taking advantage of capsaicin lipophilicity and the relatively high TRPV1 open probability evoked at saturated concentration (29), where only patches that contain a single channel were used for further analysis (Fig. 3A).…”