TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain

Abstract

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Keywords: TRPV1; chaperone; pain; progesterone; sigma 1 receptor.

Fig. 1.

TRPV1-mediated pain is dependent upon Sig-1R activity. (A) Response to 2.8-µg capsaicin injection into to the hind paw of male mice that had been pretreated for 24 h by intrascapularly injecting either vehicle (water) or a Sig-1R antagonist (BD1063) (32 mg/kg in water). PLT in response to capsaicin were 50 ± 5 s for vehicle-pretreated (n = 6) and 24 ± 4 s for BD1063-pretreated (n = 6) mice; ***P < 0.0001; ANOVA. PLT for mice injected only with vehicle or BD1063 were 19 ± 3 s and 16 ± 4 s, respectively; nonsignificant (ns), ANOVA. (B) Isolation of total protein from mouse DRGs injected 24 h before with vehicle (water) or with BD1063 (BD) (32 mg/kg) for TRPV1 and GAPDH (loading control) immunodetection. Normalized data are 69 ± 6% for BD1063 vs. control (Ctr), n = 3; **P < 0.001, Student’s t test. (C) Primary cultures of DRG neurons from mice treated with 25 µM P4 in 0.25% methanol for 24 h and from control cultures (0.25% methanol). Total protein was analyzed by Western blot (WB) for immunodetection of TRPV1 and GAPDH. The mean value for total TRPV1 protein in P4-treated cultures was 57 ± 14%; n = 4; *P < 0.05, Student’s t test.

Fig. 2.

Antagonism of Sig-1R leads to changes in TRPV1 expression in HEK293 cells. (A and B, Left) Immunodetection of TRPV1 and GAPDH total proteins from HEK293 cells expressing TRPV1 treated for 24 h with 25 µM BD1063 (BD) (A) or P4 (B) and control (Ctr) [water in A; methanol (0.25%) in B]. (A, Right) Mean values of TRPV1 for the BD group were 55 ± 6% (n = 14) with respect to control. ***P < 0.001, Student’s t test. (B, Left) Mean values of TRPV1 for P4 treatments were 65 ± 2% (n = 21) with respect to control (n = 28). **P < 0.01, Student’s t test. (C and D, Left) Plasma membrane (PM) proteins from control and 25 µM BD1063-treated cells (C) and P4-treated cells (D) were isolated for immunodetection of TRPV1 and Na⁺/K⁺-ATPase α (load control). (C, Right) PM TRPV1 mean values for BD1063 condition yielded 55 ± 9% with respect to control; (n = 3); *P < 0.05, Student’s t test. (D, Right) For P4 treatments, PM TRPV1 levels were 41 ± 5%; (n = 3); **P < 0.001, Student’s t test. Arrows point to the hyperglycosylated TRPV1 form, which decreases with BD1063 and P4 treatments. (E, Left) Average current–density plots from TRPV1-expressing HEK293 cells on the current density evoked by 4 µM capsaicin under control and BD1063-treated conditions. (Right) Averaged current density at +120 mV was 451 ± 91 pA/pF for control (water) (n = 19) and 261 ± 74 pA/pF for BD1063 treatment (n = 20). *P = 0.05; Student’s t test. (F, Left) Average current–density plots from TRPV1-expressing HEK293 cells on current density evoked by 4 µM capsaicin under control and P4-treated conditions. (Right) The averaged current density at +120 mV for control cells treated for 24 h with 0.25% methanol: 375 ± 87 pA/pF (black bar; n = 27); for control cells treated for 24 h with 0.01% methanol: 317 ± 49 pA/pF (gray bar; n = 21); for control cells treated for 24 h with 0.0001% methanol: 322 ± 85 pA/pF (light gray bar; n = 23); for cells treated for 24 h with 25 µM P4: 67 ± 17 pA/pF (dark blue bar; n = 27); for cells treated for 24 h with 134 ± 33 pA/pF (red bar; n = 23); and for cells treated for 24 h with 0.1 µM P4: 99 ± 36 pA/pF (aqua bar; n = 27). *P < 0.01 Student’s t test.

Fig. 3.

Colocalization of TRPV1 and Sig-1R in HEK293 cells and DRG neurons by confocal microscopy. (A) Panels 1 and 2 show signals detected for the TRPV1-mCherry (1, red) and Sig-1R (2, green) proteins. Panel 3 shows labeling of the ER by rabbit anti-calnexin and anti-rabbit Cy5 antibodies (blue). Colocalization of TRPV1 and Sig-1R in the ER is shown in panel 4 (arrow indicates the merge in white). (B) Panels 1 and 2 are the same as in A. Panel 3 shows the plasma membrane labelled with CellMask Deep Red (blue). Panel 4 shows the merged image (purple) and strong localization of TRPV1 (red) to the plasma membrane. (C) Colocalization of endogenous TRPV1 and Sig-1R in primary cultures of mouse DRG neurons. TRPV1 immunodetection with goat anti-TRPV1 and secondary anti-goat Alexa Fluor 488 antibodies (green, panel 1) and Sig-1R immunodetection with rabbit anti–Sig-1R and secondary anti-rabbit Alexa Fluor 594 antibodies (red, panel 2). Merge (yellow, panel 3) shows colocalization of both proteins. (Scale bars: 10 μm.) All arrows indicate colocalized signals.

Fig. 4.

TRPV1 and Sig-1R form protein–protein complexes. (A, Left) Coimmunoprecipitation of TRPV1 with Sig-1R antibody and immunodetection of TRPV1 (Upper) and Sig-1R (Lower). (Right) Coimmunoprecipitation of Sig-1R with TRPV1 antibody and immunodetection for TRPV1 (Upper) and Sig-1R (Lower). IgG served as negative control. The figure is representative of three independent experiments. Input is 1/10th of the total protein used in each immunoprecipitation. (B) Immunoprecipitation was carried out with TRPV1 antibody for the total protein from control cells (Ctr, Left) and BD1063-treated cells (Right). Differences between control and BD1063-treated cells were determined in the signal for immunoprecipitated Sig-1R (lane 5). The figure is representative of three independent experiments. (C) Immunoprecipitation was carried out as above with total protein from control cells (Left) and P4-treated cells (Right). Differences between control and P4-treated cells were determined in the signal for immunoprecipitated Sig-1R (lane 5). The figure is representative of three independent experiments. (D and E) FRET between TRPV1-mCherry and Sig-1R-GFP. The ratio A_o is the fractional excitation of mCherry in the absence of donor (GFP), and its average value is indicated by the dashed line. A value of ratio A, when both donor and acceptor are present, greater than ratio A_o is indicative of FRET. The coexpression of TRPV1 and Sig-1R produces an apparent FRET efficiency of 0.49 ± 0.02 (n = 17). (D) Incubation of the cells in the presence of 25 µM BD1063 reduces the apparent FRET efficiency to 0.42 ± 0.02 (n = 6), which is not different from the ratio A_o. (E) Treatment with 25 µM progesterone (P4) has an effect similar to BD1063. Control apparent FRET efficiency is 0.57 ± 0.03 (n = 10), and progesterone treatment reduces it to 0.45 ± 0.02 (n = 10). (F) Western blot analysis for deletion constructs of TRPV1 protein shows the bands corresponding to the expected sizes: TRPV1-ΔNH2 (58 kDa, Left), TRPV1-ΔCOOH (77 kDa, Center), and TRPV1-TM1-6 (28 kDa, Right). (G) Coimmunoprecipitation of different TRPV1 deletion constructs with Sig-1R. Coimmunoprecipitation of the TRPV1-ΔNH₂ (Top) and TRPV1-ΔCOOH (Middle) deletion constructs and of TM1-6 transmembrane domains only (Bottom) with Sig-1R. Each deletion construct of TRPV1 was coimmunoprecipitated with Sig-1R-GFP (IP) by using a specific antibody against a specific epitope of the segment.

Fig. 5.

Antagonism of Sig-1R affects TRPV1 expression and its role in pain. (A) Nonpregnant and pregnant (15 d of gestation) mice were intradermally injected into to a hind paw with vehicle- or capsaicin (2.8 µg)-containing solutions. PLTs for vehicle injections of nonpregnant (n = 18) and pregnant females (n = 12) were 5.2 ±1.2 s and 2.5 ± 0.9 s, respectively. For capsaicin injection PLTs were 43 ± 2 s in nonpregnant females and 24 ± 1 s in pregnant females. ***P < 0.0001; ns, not statistically significant; ANOVA. (B) Antagonism of Sig-1R promotes the association of Sig-1R with BiP leading to Sig-1R inactivation, protein misfolding in the ER, and, finally, to ER-associated protein degradation through the proteasome which, in turn, results in decreased TRPV1 expression levels and capsaicin-induced pain. In the absence of antagonists such as progesterone (P4) and BD1063 (BD), Sig-1R can positively regulate TRPV1 protein expression in the plasma membrane, as shown on the right side of the figure where interactions between Sig-1R and TRPV1 are not disrupted and the channel transits correctly to the Golgi apparatus (GA) where it is hyperglycosylated and then transported to the plasma membrane.