αCGRP is essential for algesic exocytotic mobilization of TRPV1 channels in peptidergic nociceptors

Abstract

Proalgesic sensitization of peripheral nociceptors in painful syndromes is a complex molecular process poorly understood that involves mobilization of thermosensory receptors to the neuronal surface. However, whether recruitment of vesicular thermoTRP channels is a general mechanism underlying sensitization of all nociceptor types or is subtype-specific remains controversial. We report that sensitization-induced Ca(2+)-dependent exocytotic insertion of transient receptor potential vanilloid 1 (TRPV1) receptors to the neuronal plasma membrane is a mechanism specifically used by peptidergic nociceptors to potentiate their excitability. Notably, we found that TRPV1 is present in large dense-core vesicles (LDCVs) that were mobilized to the neuronal surface in response to a sensitizing insult. Deletion or silencing of calcitonin-gene-related peptide alpha (αCGRP) gene expression drastically reduced proalgesic TRPV1 potentiation in peptidergic nociceptors by abrogating its Ca(2+)-dependent exocytotic recruitment. These findings uncover a context-dependent molecular mechanism of TRPV1 algesic sensitization and a previously unrecognized role of αCGRP in LDCV mobilization in peptidergic nociceptors. Furthermore, these results imply that concurrent secretion of neuropeptides and channels in peptidergic C-type nociceptors facilitates a rapid modulation of pain signaling.

Keywords: inflammation; ion channel; nociception; pain transduction; sensory neuron.

Fig. 1.

ATP-mediated TRPV1 sensitization in peptidergic nociceptors requires channel recruitment. (A and B) Representative microfluorometric recordings of ATP sensitization of capsaicin-evoked Ca⁺² influx in saporin (A) and IB4-saporin–treated nociceptors (10 nM, 72 h) (B). (C) Fold potentiation of capsaicin responses obtained as P3/P2 peak intensities. ATP (10 µM, 8 min) applied between the second (P2) and the third (P3) capsaicin pulse (200 nM, 10 s). KCl pulse (40 mM, 10 s) was applied to distinguish viable neurons. DD04107 (20 µM, a concentration that produces maximal α-CGRP inhibition without altering neuronal morphology) preincubated 1 h at 37 °C. (D and E) Representative voltage-clamp recordings of currents elicited by capsaicin (1 μM, 10 s) in IB4⁻ neurons (D) and IB4⁺ nociceptors (E). (F) Fold potentiation of TRPV1 activity. Cells held at −60 mV. TRPV1 currents were sensitized by ATP (10 µM, 8 min). Nonpalmitoylated DD04107 (100 µM) was applied through patch pipette 10 min before recording (denoted as Peptide, D, Lower). Data represent mean ± SEM, n ≥ 3 cultures, and n ≥ 100 neurons for Ca²⁺-imaging assays. For patch clamp, numbers above the bars represent the total registered neurons (n). Statistical analysis was made by one-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05; **P < 0.01.

Fig. 2.

αCGRP and Tac1 deletion decreases ATP-induced TRPV1 sensitization in nociceptors. (A) Representative microfluorometric recordings of ATP-induced potentiation of capsaicin responses in rat nociceptors transfected with Tac1 and αCGRP siRNAs (100 nM, 48 h). (B) Fold potentiation calculated as the P3/P2 ratio. (C) Representative capsaicin-induced Ca²⁺ responses in nociceptors from WT and DKO mice. (D and E) Representative microfluorometric recordings illustrating ATP sensitization of TRPV1 responses in WT and DKO nociceptors in the absence (vehicle) or presence of DD04107. (F) Fold potentiation of capsaicin-induced responses. ATP (10 µM, 8 min) was added between the P2 and the P3 pulse of capsaicin (200 nM, 10 s). KCl (40 mM) pulse was used to distinguish viable neurons. DD04107 (20 µM) was preincubated for 1 h. Data represent mean ± SEM, with n = 3, and n ≥ 100 neurons. Statistical analysis was performed by two-way ANOVA, followed by Bonferroni post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

αCGRP and Tac1 deletion decreases ATP-sensitized, TRPV1-mediated nociceptor excitability. (A) Representative MEA recordings of capsaicin-induced action potentials and desensitization (Top). Potentiation of capsaicin (500 nM, 15 s) responses was elicited by applying 10 µM of ATP between the second (P2) and third (P3) vanilloid pulse (Bottom) in WT nociceptors. (B) Representative recordings of neuronal activity of DKO nociceptors in culture using the experimental paradigm in A. (C and D) Representative recordings of ATP-induced sensitization of capsaicin-evoked neuronal excitability in WT (C) and DKO (D) nociceptors preincubated with 20 µM of DD04107 (1 h, 37 °C).

Fig. 4.

ATP-induced TRPV1 sensitization involves TRPV1 recruitment. (A) Mean spike frequency of capsaicin-induced action potential firing in WT nociceptors. (B) Mean spike frequency of vanilloid-evoked action potential firing in DKO nociceptors. (C) Fold potentiation of capsaicin-induced Ca²⁺ responses and (D) mean spike frequency of capsaicin-evoked action potential firing in WT nociceptors in the presence of 250 nM CGRP_8–37 or 10 µM CP96345 or vehicle. Capsaicin (500 nM, 15 s) and ATP (10 µM, 8 min) were used. DD04107 (20 µM) was preincubated for 1 h at 37 °C. Mean spike frequency was calculated from recordings displayed in Fig. 3. Data are expressed as mean ± SEM; n = 3 cultures. For Ca²⁺ microfluorography, n ≥ 100 neurons. For mean spike frequency, n = 30–65 electrodes. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test. For MEA, data were analyzed as paired values through comparison of the responses of each electrode in the 30-s time interval upon stimulation. ***P < 0.001; **P < 0.01; *P < 0.05. Only comparison between P2 and P3 is shown in the graphs.

Fig. 5.

TRPV1 is localized in CGRP-positive LDCVs, and ATP promotes exocytosis of TRPV1 secretory granules. (A) Immunofluorescence labeling of CGRP (red) and TRPV1 (green) distribution in mouse DRG neurons; double immunolabeling (merge) shows colocalization of TRPV1 and CGRP in LDCVs. (Scale bar 10 μm.) (Right) Pre-embedding immunogold labeling of TRPV1 is localized in the plasma membrane and in LDCVs in mouse DRG neurons. (B) Electron-microscopy images depicting immunogold labeling of TRPV1 in nociceptors treated by 10 µM ATP and/or 20 µM DD04107 as indicated. (C) Quantitation of immunogold TRPV1 particles. (D) Electron microscopy images illustrating immunogold labeling of TRPV1 in nociceptors from WT and DKO mice treated with 10 µM ATP or buffer. (E) Quantitation of immunogold TRPV1 particles in WT and DKO nociceptors. Data are expressed as mean ± SEM. One-way ANOVA followed by Bonferroni post hoc test was used. ***P < 0.001; **P < 0.01; *P < 0.05 versus vehicle-treated neurons; n = 3 cultures. Arrow indicates plasma membrane; arrowhead, LDCVs; and crossed arrow, cytoplasm. (Scale bar, 200 nm.)

Fig. 6.

ATP-induced TRPV1 sensitization requires αCGRP expression in sensory neurons. (A and B) Representative microfluorometric recordings showing sensitization of capsaicin-induced Ca²⁺ influx (300 nM, 10 s) upon incubation with ATP (10 µM, 8 min) in WT, Tac1^−/− (A), and αCGRP^−/− (B) sensory neurons. (C) Fold potentiation of capsaicin-induced Ca²⁺ responses as P3/P2 ratio. (D) Mean spike frequency of capsaicin-induced action potential firing in WT, αCGRP^−/−, and Tac1^−/− nociceptors in the presence or absence of ATP. Capsaicin (500 nM, 15 s) and ATP (10 µM, 8 min) were used. Data represent mean ± SEM, n = 3 cultures, n = 30–65 electrodes for MEA experiments, or n ≥ 100 neurons for Ca²⁺ microfluorography. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test. Data for MEA were analyzed as paired values through comparison of the responses of each electrode in the 30-s time interval upon stimulation. Only comparison between P2 and P3 is shown in the graphs. ***P < 0.001; *P < 0.05.

Fig. 7.

αCGRP expression is needed for ATP-induced thermal hyperalgesia in mice. (Left and Right) Paw withdrawal latencies to a radiant heat stimulus before (baseline) and after a 30-min intraplantar injection of 100 nmol ATP (Left) or saline (Right) in WT, αCGRP^−/−, Tac1^−/−, or DKO mice. Data represent mean ± SEM; n = 8 animals per group. Statistical analysis was performed by one-way ANOVA paired values followed by Bonferroni post hoc test; *P < 0.05; **P < 0.01 compared with baseline.