TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids

Abstract

The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels.

Figure 1. Purification of functional TRPV1 protein

(A) Rat TRPV1 protein was expressed as an amino (N)-terminal 8xHis-MBP fusion (referred to as rTRPV1 hereafter) in Sf9 cells, where capsaicin (10 μM) elicited robust channel activation as assessed by ratiometric calcium imaging. (B) Representative whole cell recording from Sf9 cells expressing rTRPV1. Capsaicin (10 μM) evoked large, outwardly rectifying current. (C) Capsaicin dose-response curve for MBP-TRPV1 expressing Sf9 cells reveals an EC₅₀ = 0.4 μM; n = 6 independent whole cell recordings. (D) rTRPV1 protein elutes predominantly as a symmetric peak within the included volume of a Sepharose 6 size exclusion column. (Inset) Affinity purified rTRPV1 protein was analyzed by SDS-PAGE (4–12% gel) and visualized by coomassie staining (~1 and 5 μg; lanes 1 and 2) or western blotting (~2 ng, 6 ng, or 18 ng; lanes 3–5) using anti-MBP antibody.

Figure 2. Functional characterization of purified TRPV1 in reconstituted soybean liposomes

(A) Patches excised from TRPV1 containing proteoliposomes showed capsaicin (cap, 10 μM)-evoked responses that were blockable by the antagonist, capsazepine (cpz, 20 μM). Current-voltage relationships showed outward rectification characteristic of TRPV1 channels. (B) Capsaicin dose-response curve for TRPV1 proteoliposomes (EC₅₀ = 10.7 μM; n = 11 independent patches). (C) Activation of TRPV1 proteoliposomes by various agonists, including 2-aminophenyl borate (2-APB, 1 mM), cap (10 μM), resiniferatoxin (RTX, 1.2 μM), and protons (pH 5 solution applied to ‘intracellular’ face of the liposome). Except for ‘intracellular’ protons, all other TRPV1 agonists produced robust responses. (D) Application of protons to the ‘extracellular’ face of proteoliposomes evoked capsazepine-blockable currents that were enhanced by addition of capsaicin (10 μM). (E) Application of spider toxin (DkTx, 2 μM) to the ‘extracellular’ face of proteoliposomes evoked capsazepine-blockable currents that were enhanced by addition of capsaicin (10 μM). (F) Schematic representation of TRPV1 orientation in ‘inside-out’ proteoliposomes patches as determined by sensitivity to protons or DkTx.

Figure 3. TRPV1 is an intrinsically heat sensitive channel

(A) Outwardly rectifying currents recorded from TRPV1-containing soybean proteoliposomes in response to a temperature ramp (22–48°C). (B–C) Thermal response profile and Arrhenius plot derived from responses in (A) reveal activation threshold (40.3°C) and temperature coefficient (Q₁₀ = 20.0) resembling that of TRPV1 channels in native biological membrane. (D) Capsaicin (5 μM)-evoked current demonstrates presence of functional TRPV1 channels in a proteoliposome patch, where subsequent response to heat (48°C) challenge was blocked by co-application of capsazepine (20 μM). (E) TRPV1 responses evoked by rapid temperature jumps delivered to proteoliposome patches using an infrared (IR) laser (V_h = −60 mV; duration of heat pulse = 100 msec). (F) Activation time course for IR laser-evoked responses fit by a single exponential (stimulus temperatures shown at right).

Figure 4. Direct activation or modulation of TRPV1 by lipid metabolites

(A–E) Thermal response profiles from TRPV1-containing proteoliposome patches in the absence or presence of inflammatory agents, including protons or lipid metabolites, as indicated. All agents potentiated TRPV1 heat sensitivity at doses below thresholds required for direct channel activation at room temperature. All agents were delivered by bath perfusion, except for protons, which were introduced in the electrode buffer (pH 6.0). Individual and averaged responses are plotted as light gray and red traces, respectively (n > 7 patches per condition). (F–G) Both anandamide (AEA) and lysophosphatidic acid (LPA) directly activated TRPV1 in proteoliposome patches. Responses evoked by various concentrations of these agonists are shown relative to capsaicin. (H) Thermal response profiles obtained from TRPV1-containing proteoliposome patches in the presence of 3 μM LPA. Individual and averaged responses are plotted as light gray and red traces, respectively (n = 7).

Figure 5. Regulation of TRPV1 by phosphoinositides

(A–B) Capsaicin (10 μM)- or heat-evoked currents were observed in minimal TRPV1-containing proteoliposomes lacking phosphoinostides. (C) With the exception of PIP₃ (4%), phosphoinositides (4%) produced rightward shift in capsaicin dose-response curve relative to minimal proteoliposomes (EC₅₀ =0.33, 0.29, 0.88, 1.36, and 1.63 μM for no phosphoinositides, PIP₃, PIP₂, PI, and PI4P, respectively). (D) Inclusion of various phosphoinositides (4%) produced marked rightward shift in thermal response profiles. TRPV1 proteoliposomes without phosphoinositides or with PIP₃ (4%) exhibit thermal response profiles characteristic of sensitized channels. Traces in (D) show averaged profiles (n > 7 patches per condition; individual traces shown in Figure S4A-G). (E) Thermal activation thresholds for curves shown in (D) Error bar denotes mean ± s.d., *p<0.0001, Student’s t-test, n > 7 per condition.

Figure 6. TRPV1 sensitivity is tuned by C-terminal- membrane interaction

(A) A C-terminally truncated TRPV1 is not inhibited in reconstituted minimal liposomes containing phosphatidyl inositol (PI, 4%). (B) Schematic depicting orthogonal chemical strategy for modulating TRPV1-lipid interactions. TRPV1 (yellow) was tagged with 8xHis (green) at the N- or C-terminus to promote interaction with head group modified lipids (DGS-NTA) in a nickel (Ni)-dependent manner. (C) Capsaicin dose-response curves for 8xHis-tagged rTRPV1 in liposomes containing DGS-NTA. C-terminal tagged channel in the presence of Ni (black) showed rightward shift (EC₅₀ = 3.69 μM; n = 8 independent patches) relative to proteoliposomes containing C-terminal fusion without Ni (blue; EC₅₀ = 1.25; n = 19 independent patches), or N-terminal fusion with Ni (red; EC₅₀ = 1.06; n = 14 independent patches) or without Ni (green; EC₅₀ = 1.20; n = 9 independent patches). (D–E) Specific interaction between TRPV1 C-terminus and membrane lipids also inhibits thermal sensitivity. Averaged or individual responses are shown as solid or dotted traces, respectively. (F) Corresponding averaged thermal activation thresholds. Error bar denotes mean ± s.d., *p<0.0001, Student’s t-test, n > 8 per condition.

Figure 7. Model depicting sensitization of TRPV1 by lipid metabolism

TRPV1 channel is inhibited by association of its C-terminus with membrane phosphoinositides (i.e, PI, PI4P, and PIP₂). Activation of PLC downstream of bradykinin or TrkA receptors by their agonists (i.e, BK or NGF, respectively) may potentiate TRPV1 through three major subsequent events: i) PLC mediated depletion of PIP₂ (as well as PI and PI4P) releases TRPV1 from negative inhibition by these lipids; ii) activated PKC phosphorylates and sensitizes TRPV1; iii) lipid metabolites such as AA and HPETE generated by PLA2 sensitize TRPV1 by directly targeting the channel. In addition, given that PIP₃ has no effect on TRPV1, on site conversion of PIP₂ to PIP₃ by PI3K may also contribute to sensitization by NGF and other factors that activate PI3K.