TRPV1 function is modulated by Cdk5-mediated phosphorylation: insights into the molecular mechanism of nociception

Abstract

TRPV1 is a polymodally activated cation channel acting as key receptor in nociceptive neurons. Its function is strongly affected by kinase-mediated phosphorylation leading to hyperalgesia and allodynia. We present behavioral and molecular data indicating that TRPV1 is strongly modulated by Cdk5-mediated phosphorylation at position threonine-407(mouse)/T406(rat). Increasing or decreasing Cdk5 activity in genetically engineered mice has severe consequences on TRPV1-mediated pain perception leading to altered capsaicin consumption and sensitivity to heat. To understand the molecular and structural/functional consequences of TRPV1 phosphorylation, we generated various rTRPV1T406 receptor variants to mimic phosphorylated or dephosphorylated receptor protein. We performed detailed functional characterization by means of electrophysiological whole-cell and single-channel recordings as well as Ca(2+)-imaging and challenged recombinant rTRPV1 receptors with capsaicin, low pH, or heat. We found that position T406 is critical for the function of TRPV1 by modulating ligand-sensitivity, activation, and desensitization kinetics as well as voltage-dependence. Based on high resolution structures of TRPV1, we discuss T406 being involved in the molecular transition pathway, its phosphorylation leading to a conformational change and influencing the gating of the receptor. Cdk5-mediated phosphorylation of T406 can be regarded as an important molecular switch modulating TRPV1-related behavior and pain sensitivity.

Figure 1. Responses of wild-type, p35KO, Cdk5CoKo, TRPV1KO, and Tgp35 mice to capsaicin and heat.

Water-deprived C57Bl6 and FVBN mice were tested for 1 h using the lickometer with a free access to water containing 15 μM capsaicin. The behavior is expressed as a % of the baseline licking responses for plain water as compared to capsaicin. Increased aversion and hypersensitivity to capsaicin was evident in Tgp35 mice (FVBN background) by decreased number of licks (unpaired t-test, p < 0.05). In contrast, p35 knockout or Cdk5CoKo mice (C57BL6/129SVJ background) showed less aversion to capsaicin compared to their littermate wild-type (WT) controls (One-way ANOVA followed by Dunnett’s multiple comparisons test, p < 0.0001). Data are presented as mean ± SEM from four animals during five different measurements (a). Effect of temperature activation of TRPV1 in mutant animals. An orofacial pain assessment device was used to measure the responses of the mice to hot facial stimulation. All mice showed similar consumption of the reward (sucrose) at 37 °C (b). Tgp35 mice displayed an aversive behavior to the increased temperature of the thermodes as noted by significantly decreased licking behavior (unpaired t-test, p = 0.0002), whereas p35KO mice displayed significantly increased number of licks compared to wild-type controls (One-way ANOVA followed by Dunnett’s multiple comparisons test, p < 0.05) (c). Data are presented as mean ± SEM from four animals measured five times using 37 °C and three times at 45 °C.

Figure 2. Cdk5-mediated phosphorylation of TRPV1 prevents desensitization to capsaicin.

TRPV1-mediated inward (−100 mV) and outward (+100 mV) currents in transiently transfected CHO cells induced by 3.3 μM capsaicin in the presence or absence of extracellular Ca²⁺. Left panels show I/V relationships corresponding to the representative recording traces on the right. In the presence of extracellular Ca²⁺, application of capsaicin for 200 s established a desensitized steady state in TRPV1 expressing cells (a,b). Co-expression of TRPV1, Cdk5-mCherry and p35-CFP inhibits the Ca²⁺-induced desensitization (c,d), similar to the capsaicin-induced currents of TRPV1 in absence of extracellular Ca²⁺ (e,f). Maximal induced currents (g), time to half-maximal response represented as t₅₀ (h) and desensitization as ratio I_steady/I_maximal (i) of n = 11–22 independent measurements. Asterisk (*) indicates significant differences compared to the corresponding TRPV1 value at Ca²⁺-containing conditions (unpaired WR-test, p < 0.05).

Figure 3. TRPV1_T406D mutants show slowed activation kinetics.

Ca²⁺-induced desensitization of TRPV1_WT and TRPV1_T406 mutants in transiently transfected CHO cells challenged with 0.3 μM or 3.3 μM capsaicin measured by voltage ramp protocols in the presence of extracellular Ca²⁺. Application of 0.3 μM (a) or 3.3 μM (b) capsaicin for 200 s induces TRPV1-mediated currents. Amplitudes of capsaicin-induced currents (c), activation kinetics represented by time to half maximum current (t₅₀) of first and second response to 0.3 μM (d) or 3.3 μM capsaicin (e). Desensitization represented as ratio (I_steady/I_maximal) of n = 8–13 independent measurements (f). Asterisk (*) indicates significant differences compared to the corresponding TRPV1_WT value (unpaired WR-test, p < 0.05).

Figure 4. Voltage-dependence of TRPV1 is altered in the T406D mutant.

Voltage-dependence of TRPV1_WT and TRPV1_T406 mutants in transiently transfected CHO cells measured by voltage step protocols with depolarizing pulses from −120 mV to +160 mV. In Ca²⁺-free ringer solution, voltage-dependent currents are detected in TRPV1_WT and TRPV1_T406A, but not in TRPV1_T406D (a). Application of 3.3 μM capsaicin induces robust voltage-dependent currents in CHO cells expressing TRPV1_WT, TRPV1_T406A or TRPV1_T406D (b). Voltage-activated currents evoked one minute after washout of capsaicin revealed an increased voltage-dependence of TRPV1_T406D, whereas the voltage-induced currents of TRPV1_WT, TRPV1_T406A recover to the same level as under Ringer’s conditions (c). Normalized conductance G/G_max of n = 6–7 independent measurements of TRPV1_WT (d), TRPV1_T406A (e), and TRPV1_T406D (f). Sigmoidal fit was used to calculate V_1/2. Asterisk (*) indicates significant decrease of V_1/2 of TRPV1_T406D after priming with 3.3 μM capsaicin (paired WR-test, p < 0.05).

Figure 5. Sensitivity of TRPV1 to capsaicin is altered in the T406D mutant.

Analysis of TRPV1 concentration/response-relationships in transiently transfected CHO cells using voltage-ramp protocols in the absence of extracellular Ca²⁺. Inward and outward currents of TRPV1_WT (a), TRPV1_T406A (b) and TRPV1_T406D (c) induced by 0.05, 0.1, 0.3, 1 and 3.3 μM capsaicin. Concentration/response-relationships of outward (d) or inward (e) currents of TRPV1_WT and TRPV1_T406A show no difference between first and second series of application. Before priming of TRPV1_T406D with 3.3 μM capsaicin, responses were small and not evaluable. Challenging cells with capsaicin (3.3 μM) led to significant increase in sensitivity and allowed the TRPV1_T406D receptor to then respond to lower concentrations (paired t-test, p < 0.05). EC₅₀ values obtained during the second series of capsaicin application (outward: TRPV1_WT 0.28 ± 0.06 μM; TRPV1_T406A 0.19 ± 0.02 μM; TRPV1_T406D 0.11 ± 0.01 μM; inward: TRPV1_WT 0.66 ± 0.14 μM; TRPV1_T406A 0.40 ± 0.07 μM; TRPV1_T406D 0.26 ± 0.03 μM). n = 6–10 independent measurements were performed for each receptor variant.

Figure 6. Sensitivity of TRPV1 to pH 6 is altered in the T406D mutant.

pH 6, 0.3 μM and 3.3 μM capsaicin-induced currents of TRPV1_WT and TRPV1_T406 mutants in the absence of extracellular Ca²⁺. Application of Ringer’s solution at pH 6 or 0.3 μM capsaicin induces currents in TRPV1_WT (a) and TRPV1_T406A (b) receptor variants. TRPV1_T406D expressing cells respond to pH 6 or 0.3 μM capsaicin only after priming the cells with 3.3 μM capsaicin (c). Ratio between first and second response indicates sensitization (<1) or desensitization (>1) during 3.3 μM capsaicin priming (d). Analysis of n = 10–15 independent measurements shows that TRPV1_T406D is sensitized by priming with 3.3 μM capsaicin (unpaired WR-test, p < 0.05).

Figure 7. Response of TRPV1 to heat is altered in the T406D mutant.

HEK293T cells expressing TRPV1_WT or TRPV1_T406D in Fura-2 Ca²⁺ imaging experiments. Representative ratio image and time course of TRPV1 n = 131 (a,b) and TRPV1_T406D n = 136 (c,d) measurements. Mean ± SEM of the TRPV1-mediated responses (Δ ratio (F₃₄₀/F₃₈₀)), induced by 42 °C or 3.3 μM capsaicin (e). Responses to first and second heat stimuli were equal in TRPV1_WT expressing cells. Compared to TRPV1_WT, the initial response of TRPV1_T406D to heat (42 °C) is significantly lower, but increases after priming with 3.3 μM capsaicin (paired t-test, p < 0.05). The activation kinetics (rise time from 10 to 90% = t_10–90) of TRPV1_T406D is significantly accelerated in the second heat-induced responses (1^st 51.1 ± 2.2 s, 2^nd 31.3 ± 0.8 s) (paired t-test, p < 0.05) (f).

Figure 8. TRPV1 single-channel properties are altered in the T406D mutant.

TRPV1 single-channel events measured in cell-attached configuration of transiently transfected CHO cells at −60 mV pipette potential (equivalent to +60 mV membrane potential). Extracellular solution with elevated K⁺ concentration was used to adjust the resting potential of the cell to 0 mV. Pipette solution contained 10 mM Ba²⁺ to silence endogenous K⁺ channels. Representative gating events of TRPV1_WT (a) and TRPV1_T406D (b) at −60 mV (i) induced by 0.3 μM capsaicin (ii) and by 0.3 μM capsaicin after 2 min incubation with 3.3 μM capsaicin (iii). Representative event distribution histograms showing the open and closed probability (NP_O/C) of TRPV1 (c) and TRPV1_T406D (d). Statistical analysis of open and close probability revealed that the open probability of TRPV1_T406D was significantly reduced at −60 mV (i), and −60 mV + 0.3 μM capsaicin, whereas the pretreatment with 3.3 μM capsaicin induces similar NP_O of TRPV1_T406D as the TRPV1_WT (e). n = 3–10 independent recordings (unpaired WR-test, p < 0.05).