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. 2009 Nov 20;284(47):32591-601.
doi: 10.1074/jbc.M109.019687. Epub 2009 Sep 24.

The endoplasmic reticulum of dorsal root ganglion neurons contains functional TRPV1 channels

Sonia Gallego-Sandín  1 , Arancha Rodríguez-García, María Teresa Alonso, Javier García-Sancho
Affiliations

The endoplasmic reticulum of dorsal root ganglion neurons contains functional TRPV1 channels

Sonia Gallego-Sandín et al. J Biol Chem. .

Abstract

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca(2+) channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1(ER)). We have studied the properties of TRPV1(ER) in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1(ER) with capsaicin or other vanilloids produced an increase of cytosolic Ca(2+) due to Ca(2+) release from the ER. The decrease of [Ca(2+)](ER) was directly revealed by an ER-targeted aequorin Ca(2+) probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1(ER) to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1(ER) was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca(2+) because the sensitivity to capsaicin was increased by loading the cells with the Ca(2+) chelator BAPTA. Decreasing [Ca(2+)](ER) did not affect the sensitivity of TRPV1(ER) to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Delta35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1(ER) for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatidylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1(ER) to agonists may be critical for neuron health, because otherwise Ca(2+) depletion of ER could lead to ER stress, unfolding protein response, and cell death.

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Figures

FIGURE 1.
FIGURE 1.
Effects of activation of endogenous TRPV1 channels on Ca2+ release from the ER in DRG neurons. A, expression of TRPV1 revealed by TRPV1 antibody (see “Experimental Procedures”). Two different z sections of the same neuron are shown. B, effects of stimulation with capsaicin (CAPS; 20 μm) on [Ca2+]C in fura-2-loaded cells. In order to avoid Ca2+ entry, the stimulation with capsaicin was performed in Ca2+-free medium containing 10 μm ruthenium red (EGTA). The effects of depolarization with high K+ solution (70 mm; K+) and stimulation with caffeine (50 mm; CAF) are also shown for comparison. C, effects of stimulation with capsaicin (20 μm) on [Ca2+]ER. DRGs were infected with the HSV-ermutGA amplicon virus, and aequorin was reconstituted with coelenterazine n in Ca2+-free medium prior to the experiment. Cells were permeabilized with 20 μm digitonin in intracellular-like Ca2+-free medium (No Ca2+), and then 100 nm Ca2+ (buffered with EGTA) was added, followed by capsaicin (CAPS; 20 μm) or caffeine (CAF; 50 mm), as shown.
FIGURE 2.
FIGURE 2.
Co-localization of TRPV1 with an ER marker in HeLa (A–H) and HEK293T (I–L) cells. Cells were co-transfected with GFP-TRPV1 and erRA as described under “Experimental Procedures.” A–D, comparison of the expression of both proteins in a HeLa cell. From left to right, GFP-TRPV1 (A), erRA (B), merge image (C), and TRPV1/ER ratio (D; pseudocolor-coded, scale at right). E–H, a more equatorial section of the same cell as in the top row. I–L, co-expression in HEK293T cells. The arrows indicate areas where TRPV1 expression predominates over ER expression.
FIGURE 3.
FIGURE 3.
Capsaicin-induced Ca2+ release from the ER in TRPV1-expressing HEK293T cells. The effects of different concentrations of capsacin (CAPS; followed by concentration in μm) are shown. The ER release is evidenced by either the increase in [Ca2+]C in cells expressing cytosolic aequorin (A), the decrease of [Ca2+]ER in intact cells expressing ER-targeted aequorin (B), or the decrease of [Ca2+]ER in digitonin-permeabilized cells expressing ER-targeted aequorin (C). Ruthenium red was used to avoid entry of Ca2+ through plasma membrane-located TRPV1 in intact cells (A and B). Permeabilization with digitonin in C was performed as in Fig. 1C, except that the concentration of digitonin was 60 μm.
FIGURE 4.
FIGURE 4.
Effects of different TRPV1 channel agonists on Ca2+ release from the ER in TRPV1-transfected HEK293T cells. A, intact cells stimulated with either olvanil, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), or capsaicin (CAPS). B, digitonin-permeabilized cells stimulated with 2 μm resiniferatoxin (RTX). Details are as in Fig. 3C.
FIGURE 5.
FIGURE 5.
Concentration dependence of the effects of capsaicin on the release of Ca2+ from the ER in TRPV1-expressing HEK293T cells. The ER release was evidenced by either the increase in [Ca2+]C in cells expressing cytosolic aequorin (A), the decrease of [Ca2+]ER in intact cells expressing ER-targeted aequorin (B), or the decrease of [Ca2+]ER in digitonin-permeabilized cells expressing ER-targeted aequorin ([Ca2+]C maintained at 100 nm by a EGTA/Ca2+ buffer) (C). The extracellular medium contained 10 μm ruthenium red in A and B. Other details were as in Fig. 3. D, dose-response curve. Data were taken from A (Δ[Ca2+]C, inverted triangles), B (−Δ[Ca2+]ER; circles) and C (−Δ[Ca2+]ER; triangles) and were normalized as percentage of the maximum effect.
FIGURE 6.
FIGURE 6.
Effects of preloading with BAPTA on capsaicin-induced Ca2+ release from the ER in TRPV1-expressing HEK293T cells. BAPTA was loaded into the cytosol by incubation of the cells with 10 μm BAPTA, acetoxymethyl ester for 60 min at 20 °C, and then the ER was allowed to refill with Ca2+, and cells were stimulated with low capsaicin concentrations (CAPS; 1 or 2 μm). The results obtained in control (A) and BAPTA-loaded cells (B) are compared. Ruthenium red was added to prevent entry of Ca2+ from the extracellular medium through the plasma membrane-located TRPV1 channels. C, means ± S.E. of six similar experiments. **, p < 0.01; ***, p < 0.001 (Student's t test).
FIGURE 7.
FIGURE 7.
Capsaicin-induced release of Ca2+ from the ER at very low [Ca2+]ER in TRPV1-expressing HEK293T cells. Cells were transfected with ER-targeted native aequorin. After permeabilization with 60 μm digitonin in Ca2+-free medium, refilling of the ER was effected at low [Ca2+]C (20 nm). Consequently, the refilling proceeded to steady-state [Ca2+]ER levels much lower than when the usual refilling (at 100 nm [Ca2+]C) was performed (compare with Figs. 3C, 4A, and 6). Stimulation with capsaicin (CAPS; 1, 2, or 20 μm) or inositol trisphospate (IP3) was as shown.
FIGURE 8.
FIGURE 8.
Capsaicin-induced release of Ca2+ from the ER in HEK293T cells expressing different TRPV1 mutants. A, comparison of the Ca2+ release induced by capsaicin on the native TRPV1 (in black) and the mutants K155A, Δ35AA, and K155A/Δ35AA (different gray levels, as shown), all tested at 1 μm capsaicin. The empty plasmid vector, the control mutant R181A, or the unglycosylated N604S mutant did not increase the affinity for capsaicin (not shown). B, comparison of the dose response curves of the control (black) and the K155A mutant (gray). Shown are the average values of 3–14 determinations. Bars, S.E. C, stimulation by 1 μm capsaicin in K155A is antagonized by 10 μm capsazepine.
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