Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3

Abstract

Thermal transient receptor potential (TRP) channels, a group of ion channels from the transient receptor potential family, play important functions in pain and thermal sensation. These channels are directly activated by temperature and possess strong temperature dependence. Furthermore, their temperature sensitivity can be highly dynamic and use-dependent. For example, the vanilloid receptor transient receptor potential 3 (TRPV3), which has been implicated as a warmth detector, becomes responsive to warm temperatures only after intensive stimulation. Upon initial activation, the channel exhibits a high-temperature threshold in the noxious temperature range above 50 °C. This use dependence of heat sensitivity thus provides a mechanism for sensitization of thermal channels. However, how the channels acquire the use dependence remains unknown. Here, by comparative studies of chimeric channels between use-dependent and use-independent homologs, we have determined the molecular basis that underlies the use dependence of temperature sensitivity of TRPV3. Remarkably, the restoration of a single residue that is apparently missing in the use-dependent homologs could largely eliminate the use dependence of heat sensitivity of TRPV3. The location of the region suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain.

Keywords: TRP channel; hyperalgesia; hysteresis; thermoreceptor; use dependence.

Fig. 1.

Use dependence of wild-type TRPV3. (A) Heat responses evoked by a series of identical temperature jumps (53 °C). Each temperature pulse was 100 ms. (Inset) Enlarged view of a single pulse response. (B) Statistical plot of fold increase of peak response over repetitive stimulation. Data were normalized to the first pulse response (n = 7). (C) Responses to a family of temperature jumps ranging from 32–59 °C for the first round of activation (Left) and for repeated activation in the same patch (Right). (D) Temperature (T)-dependent responsiveness curves. Data from 10 independent experiments are superimposed. (E) van’t Hoff plots of current responses for determination of energetics. Linear fittings correspond to ΔH = 86 ± 6 kcal/mol (n = 10) for the first run and ΔH = 32 ± 1 kcal/mol (n = 10) for the repeated run. The upper portion of the plot for the second run has a shallower slope, corresponding to ΔH = 18 ± 1 kcal/mol (n = 10). The holding potential was −60 mV. I/Imax, normalized current to the maximum at 59 °C.

Fig. 2.

N-terminal MPD mediates use dependence. (A) Responses of the resulting chimera to repetitive temperature pulses (53 °C). (B) Average plot of fold increase of peak response with respect to repetition of stimulation (n = 11). (C) Responses to a family of temperature jumps in two consecutive runs. (D) Comparison of temperature-dependent responsiveness curves between initial and repeated activations (n = 10). (E) Comparison of energetics (Left, n = 10) and maximum current responses (Right, n = 10). The relative change of the response at 59 °C was plotted. The holding potential was −60 mV.

Fig. 3.

Identification of molecular regions underlying use dependence of heat sensitivity. (A) Sequence alignment of the N-terminal MPD between TRPV3 and TRPV1. Identical residues are highlighted in black. (B) Schematic diagrams showing subregions within the MPD that were exchanged between TRPV3 and TRPV1. Residue numbering is according to mouse TRPV3. (C and D) Representative chimeric responses of TRPV3 evoked by repetitive temperature pulses (53 °C). Chimeras containing exchange of region 410–414 or greater resulted in stable activity. Means of 10–11 independent recordings, normalized to sensitized responses, are shown for each average plot. (E and F) Representative chimeric responses evoked by a family of temperature jumps applied consecutively (30–59 °C). Average plots were each from 10 to 11 experiments. (G) Energetics of representative chimeras. (H) Relative change of the maximum current response. The holding potential was −60 mV.

Fig. 4.

Loop region 412–414 (404–407 in TRPV1) controls use dependence in TRPV3. (A) Structural diagram for illustration of the loop and its adjacent elements (Protein Data Bank ID code 3J5P). MPD is shown in red, and ankyrin repeats are shown in orange. (B) Replacement of the loop in TRPV3 abolished use-dependent sensitization evoked by repetitive stimulation. The fold increase relative to the initial response was shown for the average plot (n = 11). (C) Time-resolved activation by a family of temperature jumps from 31 to 57 °C [initial activation (Left) and repeated run (Right)]. (D) Comparison of temperature-dependent response curves between runs (n = 10). (E) Enthalpy changes for the initial and repeated activations (Left, n = 10) and relative change of the maximum response (Right, 57 °C). The holding potential was −60 mV.

Fig. 5.

Identification of critical residues in the loop region. (A) Insertion of a serine residue at position 412 (412S) resulted in a relatively stable heat response over repetitive stimulation. (B) Average plot for fold change of current at each repetition relative to the initial response (n = 10). (C) Responses of the mutant (412S) evoked by a family of temperature jumps (31–57 °C). (D) Temperature-dependent response curves of the 412S mutant (black for the first run and red for the second run). (E and F) Temperature-dependent response curves for mutants containing replacement of residues at other locations on the loop (black for the first run and red for the second run). (G) Summary plot of ΔH of mutant channels. The holding potential was −60 mV.

Fig. 6.

Influence of side-chain structure on use dependence. (A and B) Activation time courses and temperature responsiveness curves for the most effective insertion of valine at position 412. Averages from 10 to 12 independent recordings were plotted (the same below). (C–F) Temperature responsiveness curves for insertions with other residues (Thr, Gly, Ala, and Asn) at position 412. (G) Summary plot of ΔH (n = 10–12). The holding potential was −60 mV.

Fig. S1.

Heat responses of wild-type TRPV3 at +60 mV. (A) Heat-evoked currents by repetitive temperature pulses at 53 °C. Each temperature pulse was 100 ms, and the interpulse duration was 5–10 s. (B) Average plot of relative changes in peak current with respect to repetition of stimulation (n = 5). (C) Heat responses to a family of temperature jumps [first run (Left) and second run (Right)]. (D) Comparison of current–temperature relationships between two consecutive runs. (E) Quantitative analyses of temperature dependence. The slope of the linear fit on the lower portion of the curve provides ΔH = 86 ± 1.2 kcal/mol for initial activation and ΔH = 25 ± 1.4 kcal/mol for repeated activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV. I/Imax, normalized current to the maximum at 57 °C; T, temperature.

Fig. S2.

Heat responses of chimera TRPV3/V1(412–414) at +60 mV. (A) Heat-evoked currents by repetitive temperature pulses at 53 °C. (B) Relative changes of responses with respect to repetition of stimulation, averaged from n = 5 independent recordings. (C) Heat responses to repeated applications of a family of temperature jumps [first run (Left) and second run (Right)]. (D and E) Comparison of temperature dependence of heat responses between two consecutive runs of activations. The lower linear portions of the plots in E have slopes corresponding to ΔH = 27 ± 0.4 kcal/mol for the first run of activation and ΔH= 24 ± 0.2 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

Fig. S3.

Heat responses of mutant TRPV3-412S at +60 mV. (A) Heat-evoked currents by repetitive stimulation with a temperature pulse of 53 °C. (B) Relative changes of responses with respect to repetition of stimulation (n = 5). (C) Heat responses to repeated applications of a family of temperature jumps [first run (Left) and second run (Right)]. (D and E) Comparison of temperature dependence of heat responses between two runs. The lower linear portions of the plots in E have slopes corresponding to ΔH = 27 ± 0.6 kcal/mol for the first run of activation and ΔH= 23 ± 0.2 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

Fig. S4.

Heat responses of mutant TRPV3-412V at +60 mV. (A) Heat-evoked currents by repetitive stimulation at 53 °C. (B) Relative changes of currents over the course of repetition of stimulation (n = 5). (C) Heat responses to repeated applications of a family of temperature jumps [initial stimulation (Left) and repeated stimulation (Right)]. (D and E) Comparison of temperature dependence of heat responses between two consecutive runs of activations. The lower linear portions of the plots in E have slopes corresponding to ΔH = 25 ± 0.7 kcal/mol for the first run of activation and ΔH = 25 ± 1 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

Fig. S5.

Voltage sensitivity of wild-type mutant TRPV3 channels. (A and B) Responses of wild-type TRPV3 evoked by voltage pulses (+140 mV) were stable over repetitive stimulation. Each voltage pulse was 100 ms long. The average value represents the mean of n = 5 independent recordings. (C–E) Responses of wild-type TRPV3 evoked by voltage steps from −140 mV to +160 mV (with an increment of 20 mV) were also stable between repetitions. Current–voltage relationships were overlapping (D), whereas the maximum current at +160 mV stayed comparable (E). Data points were averages of n = 6 independent patches. Parallel plots for chimera and mutant TRPV3 channels [TRPV3/V1(412–414) (F and G), TRPV3-412S (H and I), and TRPV3-412V (J and K)]. Analyses were based on n = 5 independent experiments. Recordings were all from transiently transfected HEK293 cells at the ambient temperature. Vm, membrane potential.

Fig. S6.

Control experiments showing effectiveness of polylysine (PLL) for screening membrane PIP₂. (A) Addition of 0.01% PLL in pipette abolished responses of TRPM8 evoked by menthol (100 μM). (B) Average plot of inhibition of TRPM8 by PLL (n = 6). Currents were normalized to their initial responses after breaking in. Recordings were all from transiently transfected HEK293 cells at the ambient temperature. The holding potential is −60 mV.

Fig. S7.

Use dependence of TRPV3 remains after screening of membrane PIP₂ by polylysine. (A and B) Heat responses evoked by temperature pulses at 53 °C (100 ms) were still sensitized by repetitive stimulation in the presence of polylysine (n = 5). (C) Activations by a family of temperature pulses continued to have distinct profiles between repetitions. Experiments used fewer and more moderate temperature pulses to help maintain patch stability. (D and E) Comparison of temperature dependence of heat responses between initial and repeated activations. Linear slopes of the plots in E resulted in estimates of ΔH = 86 ± 5 kcal/mol for initial activation and 28 ± 1 kcal/mol for repeated activation (n = 8). Recordings were from transiently transfected HEK293 cells. The holding potential is −60 mV. Polylysine (0.01%) was applied through a patch pipette.

Fig. S8.

Use dependence of TRPV3 remained after receptor-mediated depletion of membrane PIP₂. (A and B) Control experiments showing effectiveness of rat M1-mediated depletion of membrane PIP₂. Persistent stimulation of rat M1 by carbachol (CCH; 30 μM) in cells coexpressing rat M1 and TRPM8 resulted in sustained inhibition of menthol (100 μM) responses. Data shown were the average of n = 6 independent experiments. (C–E) Heat responses of TRPV3 following PIP₂ depletion. TRPV3 was coexpressed with rat M1 receptors and was activated, whereas rat M1 was stimulated by CCH. Responses remained sensitized during repeated activations, with changes in time course, activation threshold, and slope sensitivity resembling those changes under control conditions. Linear fitting of slopes in E resulted in ΔH = 67 ± 5 kcal/mol for initial activation and 24 ± 0.2 kcal/mol for repeated activation (n = 6). Recordings were from transiently transfected HEK293 cells. The holding potential is −60 mV.

Fig. S9.

Simulation of allosteric models. (A) Simulated dose–response curves from an allosteric model showing that the increase of the allosteric coupling factor has opposite effects on the midpoint and slope sensitivity of the curve; that is, it decreases the midpoint while increasing the slope. Simulation was based on a MWC (Monod–Wyman–Changeux) model, Po = 1/(1 + L⁻¹ [(1 + [Ca²⁺]/K_d)/(1 + c * [Ca²⁺]/K_d)]⁴), where L is intrinsic opening and c is allosteric coupling, with parameters adapted from the parameters of BK (big potassium) channels (K_d = 32 μM, L = 7.9 × 10⁻³, and c = 2.4 or 24). (B) Heat responsiveness curves of wild-type TRPV3 fitted by an allosteric model. The black line shows the fit for initial responses upon the first round of activation. The red line represents the best fit for responses upon the second round of activation while holding temperature sensor properties (J₀ and ΔH as below) the same as for the fit in the first round of activation. The model response was calculated by I = I₀ * exp(ΔH_i/RT)/[1 + L⁻¹(1 + J)/(1 + cJ)], where I₀ * exp(ΔH_i/RT) describes the maximum current and its temperature dependence (ΔH_i was fixed to 4 kcal/mol), and J = J₀exp(ΔH/RT) describes temperature sensor equilibrium (first fit: L = 1.15 × 10⁻³, c = 4,303, ΔH = 90 kcal/mol, J₀ = 2.85 × 10⁻⁵⁹, and I₀ = 1; second fit: L = 1.1e-3, c = 1.4 × 10⁶, ΔH = 90 kcal/mol, and I₀ = 0.35).

Fig. S10.

Cryo-EM structures of TRPV1 in the identified loop region (colored in red). Closed [Left, PDB (Protein Data Bank) ID code 5irx] and open (Right, PDB ID code 5irz) cryo-EM structures are shown. Residue S404 is the critical serine residue missing in TRPV3. The residue is in proximity to the S2–S3 linker (colored in orange), with distances to V508 (closed: 4.48 A; open: 3.93A) and D509 (closed: 4.73A; open: 4.4A) in the range for van der Waals interactions. The lower side of the loop interfaces with the last two ankyrin repeats (colored in yellow), where residue T406 is H-bonded to S342 in the closed structure and P407 is H-bonded G344 in the open structure (H-bonds shown in green).