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.

Keywords: electrophysiology; gating; site-directed mutagenesis; structure-function; transient receptor potential channels (TRP channels).

FIGURE 1.

Substitutions 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).

FIGURE 2.

rTRPV1 Tyr⁵¹¹ substitutions exhibit impaired capsaicin sensitivity. A, current traces of whole cell recordings from HEK293T cells expressing the wt rTRPV1 (top panel), and the mutants rTRPV1 containing the Y511W (middle panel), and Y511M (bottom panel) substitutions at a holding potential of −40 mV. Cells were first exposed to pH 5.5 (empty bars; as a reference for channel expression) followed by applications of 1 and 30 μm capsaicin (Cap; gray and black bars, respectively). Bars above the trace indicate the time course of each activator application. B, mean/scatter-dot plot representing the normalized amplitude of whole cell currents in cells expressing rTRPV1 with indicated Tyr⁵¹¹ substitutions, evoked by 1 (gray triangles) or 30 μm (black circles) capsaicin, normalized to the current amplitude of the pH 5.5 response. Statistical significance between responses to different capsaicin concentrations for each construct was determined with paired Student's t test, where *** represents p ≤ 0.001 and ns represents a non-significant difference (n = 5–9 cells). Note a reduction in the capsaicin-evoked response in all tested mutants. Importantly, the recordings from Y511M and Y511I constructs could only be obtained when overexpressed. C, normalized concentration-response relationships to protons of wt and the indicated mutant receptors. Points represent the mean ± S.E. response of 6–7 HEK293T cells and solid lines are fit to the Hill equation: rTRPV1 (full circles, black line; n_H = 1.1 ± 0.2; EC₅₀ = pH 5.7 ± 0.3), rTRPV1 (Y511W) (empty triangle, dark gray line; n_H = 1.1 ± 0.1; EC₅₀ = pH 5.9 ± 0.1), and rTRPV1 (Y511F) (empty circle, light gray line; n_H = 0.9 ± 0.1; EC₅₀ = pH 5.8 ± 0.3). Holding potential of −40 mV.

FIGURE 3.

Substitutions of rTRPV1 Tyr⁵¹¹ to aromatic amino acids lead to a parallel rightward shift in capsaicin dose-response. A, current traces of whole cell recordings from HEK293T cells expressing either wt (rTRPV1; top panel), or mutant (rTRPV1 Y511F; bottom panel) receptor at holding potential of −40 mV. Cells were exposed to increasing concentrations of capsaicin (Cap) as indicated. Empty bars above the trace indicate the time course of each concentration application. B, normalized capsaicin concentration-response relationships of wt and the indicated mutated receptors. Points represent the mean (± S.E.) response of 6–9 HEK293T cells and solid lines are fit to the Hill equation: rTRPV1 (full circles, black line; n_H = 1.2 ± 0.1; EC₅₀ = 0.17 ± 0.02 μm), rTRPV1 (Y511W) (empty triangle, dark gray line; n_H = 1.7 ± 0.1; EC₅₀ = 1.40 ± 0.04 μm), and rTRPV1 (Y511F) (empty circle, light gray line; n_H = 1.5 ± 0.1; EC₅₀ = 1.98 ± 0.08 μm). Holding potential of −40 mV.

FIGURE 4.

A maximal open-channel lifetime is achieved when an aromatic residue occupies position 511. A, representative current traces from outside-out patches of capsaicin-exposed Flp-in T-REx HEK293 cells transiently expressing the indicated constructs. Upward (outward) currents indicate channel opening (gray dash line). Shown are representative channel activities upon exposing the patches to rTRPV1-saturating (1 μm; light gray bar; left panel) and maximal (30 μm; dark gray; right panel) capsaicin concentrations. Holding potential at +50 mV were sampled at 50 kHz and filtered at 1 kHz for display. B, mean/scatter-dot plot (n = 4–8 patches) representing the open probability (top panel, P_open) and amplitude (bottom panel) of the indicated rTRPV1 single-channel currents activated by 1 or 30 μm capsaicin. Statistical significance between the wt and mutants rTRPV1 in each capsaicin concentration are indicted as ***, p ≤ 0.001 (ANOVA followed by multiple comparison test). ND, not determined due to lack of activity.

FIGURE 5.

Substitutions of the Tyr⁵¹¹ increase the current washout rate. A, superimposed normalized whole cell washout currents of indicated constructs (wt rTRPV1, black line; rTRPV1 (Y511F), dark gray line; rTRPV1 (Y511W), light gray line) exposed to 30 μm capsaicin (Cap). Inset, superimposed normalized whole cell activation currents of the same constructs. Holding potential was −40 mV. B, mean/scatter-dot plot (n = 5–7 cells) representing the rate (τ) of activation (left) and washout (right) of the whole cell currents from the indicated constructs evoked by 30 μm capsaicin. Rates were determined by exponential fitting of the data. Statistical significances between the wt and mutants rTRPV1 are indicted as ***, p ≤ 0.001 (ANOVA followed by multiple comparison test).

FIGURE 6.

Sensitivity and washout rate of the endovanilloid NADA are affected by Tyr⁵¹¹ substitutions. A, box and whiskers plot shows NADA (2 μm)-evoked calcium response of HEK293T cells expressing rTRPV1 with or without the indicated Tyr⁵¹¹ substitutions, normalized to the response to 2-APB (0.3 mm). Boxes are mean of 3 independent experiments (each n ≥ 50 cells). Statistical significance between normalized responses of wt and mutated rTRPV1 are indicted as **, p ≤ 0.01 and ***, p ≤ 0.001 (ANOVA followed by a multiple comparison test). B, mean/scatter-dot plot (n = 6–7 cells) representing normalized amplitudes of the whole cell currents in cells expressing rTRPV1 with or without the indicated Tyr⁵¹¹ substitutions, evoked by 2 μm NADA, normalized to the current amplitude evoked by pH 5.5. Statistical significance between normalized responses of wt rTRPV1 and different mutant constructs is indicted as ***, p ≤ 0.001 (ANOVA followed by multiple comparison test). C, superimposed normalized whole cell washout currents of the indicated constructs (wt rTRPV1, black line; rTRPV1 (Y511F), dark gray line; rTRPV1 (Y511W), light gray line) exposed to 2 μm NADA. Holding potential at −40 mV. D, mean/scatter-dot plot (n = 4–7 cells) representing the washout rate (τ) of whole cell currents evoked by 2 μm NADA. Rates were determined by exponential fitting of the data. Statistical significances between the wt and mutants rTRPV1 are indicted as **, p ≤ 0.01 (ANOVA followed by multiple comparison test).