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J Biol Chem. 2008 July 11; 283(28): 19540–19550. doi: 10.1074/jbc.M707865200. | PMCID: PMC2443658 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor
Potential Vanilloid 1
Sensitization *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Irina Vetter,‡ Wei Cheng,§ Madusha Peiris,‡ Bruce D. Wyse,‡ Sarah J. Roberts-Thomson,‡ Jie Zheng,§ Gregory R. Monteith,‡ and Peter J. Cabot‡1 ‡School of Pharmacy, The University of Queensland, St Lucia, Queensland 4072, Australia and the §Department of Physiology and Membrane Biology, School of Medicine, The University of California, Davis, California 95616 Received September 20, 2007; Revised May 8, 2008. TRPV1 is a nociceptive, Ca2+-selective ion channel involved in
the development of several painful conditions. Sensitization of TRPV1
responses by cAMP-dependent PKA crucially contributes to the development of
inflammatory hyperalgesia. However, the pathways involved in potentiation of
TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells
stably expressing TRPV1 and the μ opioid receptor, we demonstrated that
treatment with the adenylate cyclase activator forskolin significantly
increased the multimeric TRPV1 species. Pretreatment with the μ opioid
receptor agonist morphine reversed this increased TRPV1 multimerization. FRET
analysis revealed that treatment with forskolin did not cause multimerization
of pre-existing TRPV1 monomers on the plasma membrane and that intracellular
pools of TRPV1 exist mostly as monomers in this model. This suggests that
increased TRPV1 multimerization occurred from an intracellular store of
inactive TRPV1 monomers. Treatment with forskolin also caused an increase in
TRPV1 expression on the plasma membrane not resulting from increased TRPV1
expression, and this rapid TRPV1 translocation was inhibited by treatment with
morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves
plasma membrane insertion of functional TRPV1 multimers formed from an
intracellular store of inactive TRPV1 monomers. This potentiation occurs
rapidly and can be dynamically modulated by activation of the μ opioid
receptor under conditions where cAMP levels are raised, such as with
inflammation. Increased translocation and multimerization of TRPV1 channels
provide a cellular mechanism for finetuning of nociceptive responses that
allow for rapid modulation of TRPV1 responses independent of transcriptional
changes. The ability to sensitize or desensitize painful stimuli is fundamental for
survival. In inflammation, sensitization of peripheral nociception contributes
to the development of hyperalgesia. Pro-inflammatory mediators including
prostaglandins mediate an increase in cellular cAMP levels, which in turn
leads to sensitization of nociception as a result of activation of
cAMP-dependent protein kinase
(PKA) 2
( 1). Such sensitization may
involve the transient receptor potential vanilloid 1 (TRPV1)
( 2). TRPV1 is a calcium-permeable ion channel that is activated by the
prototypical agonist capsaicin, the component that conveys the sensation of
“heat” to chili peppers
( 3). Endogenous TRPV1
activators include lipophilic arachidonic acid metabolites such as
N-arachidonoyl-dopamine and 12-hydroperoxyeicosatetraenoic acid
( 4,
5), but also heat and protons
( 3). TRPV1 and endovanilloid
signaling are implicated in various inflammatory hyperalgesic conditions. The
contribution of TRPV1 to inflammatory hyperalgesia has been established
through observations that TRPV1 antagonists can dose-dependently reverse both
thermal and mechanical inflammatory hyperalgesia
( 4,
5). In addition, thermal
inflammatory hyperalgesia is significantly reduced in TRPV1 knock-out mice
( 6). Several pro-inflammatory mediators and cytokines, including prostaglandin
E2, sensitize TRPV1 responses under inflammatory conditions
( 7). Moreover, the TRPV1 is
sensitized directly by cAMP-dependent PKA
( 8), making the TRPV1 a direct
molecular target for the development of inflammatory hyperalgesia
( 2,
9). Although potentiation of
TRPV1 responses by cAMP-dependent PKA appears crucial in the development of
inflammatory hyperalgesia, the mechanisms underlying this potentiation are not
fully understood. Peripheral sensitization of nociceptive responses represents a cellular
process to encourage rest and recovery after injury. The ability to modify
responses rapidly, without having to rely on altering expression levels of
nociceptive receptors, is essential for rapid adaption to external
circumstances. This system is exemplified by the release of endogenous opioids
from immune cells, which affects analgesia in inflamed tissues
( 10), and the increased
effectiveness of exogenous opioids in inflammation
( 11). The capacity for rapid
fine-tuning and modulation of sensitized nociception is further demonstrated
by the ability of opioids to inhibit TRPV1-mediated capsaicin responses
potentiated by cAMP-dependent PKA
( 12). To date, no mechanism
has been proposed to explain how TRPV1 sensitization by cAMP-dependent PKA can
be achieved in a manner that provides for rapid potentiation while maintaining
the capacity for dynamic modulation. Here, we present evidence for a model that describes how, as a consequence
of activation of adenylate cyclase, an intracellular pool of inactive TRPV1
monomers is transported to the plasma membrane where they function as TRPV1
multimers. This mechanism incorporates 2-fold regulation involving both
altered multimerization and trafficking. It provides the basis for rapid
modulation of nociceptive TRPV1 responses and finetuning of nociception.
Modification of this pathway by the opioid receptor agonist morphine can
rapidly alter TRPV1 potentiation and thus allows dynamic regulation of
nociceptive TRPV1 responses. Microplate Reader Measurement of Intracellular
Ca2+ Responses in TRPV1/MOP HEK
Cells—Double stable HEK293 cells expressing the μ opioid
receptor (MOP) and TRPV1 (TRPV1/MOP HEK cells) were generated as described
( 12). TRPV1/MOP HEK cells were
plated on PDL-coated 96-well plates and loaded with the fluorescent calcium
probe Fluo-3-AM (6 μ m) as described
( 12). Cells were washed
2–3 times with physiological salt solution (PSS; pH 7.4, composition KCl
5.9 m m, MgCl 2 1.5 m m,
NaH 2PO 4 1.2 m m, NaHCO 3 5.0
m m, NaCl 140.0 m m, glucose 11.5 m m,
CaCl 2 1.8 m m, and HEPES 10.0 m m) and
incubated for 15–30 min with pretreatments as appropriate. Preincubation
steps and Ca 2+ measurement were carried out at 29 °C to avoid
dye sequestration. Changes in fluorescence after addition of capsaicin were measured using a
fluorescent multi-well plate reader (NOVOstar, BMG Labtechnologies, Victoria,
Australia) with the excitation wavelength set at 485 nm and emission recorded
at 520 nm. As previously described, calcium responses were represented as
ΔF/F values ( 12).
Maximum ΔF/F values were used to fit a 4-parameter logistic Hill
equation to the data using GraphPad Prism (San Diego, CA, Version 4.03) to
generate dose-response curves. All experiments were designed to include
control experiments on the same plate as treated cells. For calcium measurements using the FLIPRTETRA fluorometric
imaging plate reader (Molecular Devices, Sunnyvale, CA), TRPV1/MOP HEK cells
were plated on PDL-coated, black-walled 96-well plates (Corning, Lindfield,
NSW, Australia). After loading with Fluo-3 AM (6 μm), cells were
washed three times with PSS or nominal calcium-free PSS (composition: KCl 5.9
mm, MgCl2 1.5 mm,
NaH2PO4 1.2 mm, NaHCO3 5.0
mm, NaCl 140.0 mm, glucose 11.5 mm, and HEPES
10.0 mm) as appropriate. Capsaicin and other reagents were injected
from 3–4× concentrated stock solutions prepared in PSS or nominal
calcium-free PSS with maximum final ethanol concentrations not exceeding
0.003%. Fluo-3-loaded cells were excited at 470–495 nm, and emission at
515–575 nm was recorded every second using a cooled CCD camera. For
experiments requiring no extracellular calcium, BAPTA (final concentration 100
μm) was added 30 s prior to addition of capsaicin. Whole Cell cAMP Accumulation Assay—TRPV1/MOP HEK cells were
harvested with Versene (Invitrogen, Mount Waverley, Victoria, Australia) to
avoid internalization of receptors. Cells were resuspended with anti-cAMP
acceptor bead mix yielding final concentrations of 40,000 cells/well and 1
unit/well of anti-cAMP acceptor beads. Cells were incubated for 15–30
min with agonist in triplicate on 384-well plates (OptiPlate-384; PerkinElmer
Life Sciences, Rowville, Victoria, Australia) and the reaction terminated by
addition of lysis buffer containing Streptavidin Donor beads (1 unit/well) and
biotinylated cAMP (1 unit/well). The plate was incubated under low light
conditions for 16 h, and the bioluminescence reaction measured using the
Envision Multilabel Plate Reader (PerkinElmer Life Sciences). SDS-PAGE and Western Blotting—To assess monomeric and
multimeric TRPV1 species, total cell protein was isolated and separated by
SDS-PAGE essentially as previously described
( 13). Cells were grown to
~80% confluency and incubated for 25–30 min at 37 °C with the
appropriate treatments in PSS. Cells were then immediately placed on ice,
washed with ice-cold phosphate-buffered saline (PBS; composition in
m m: NaCl 137, KCl 2.7, NaH 2PO 4 10,
KH 2PO 4 1.8) and dislodged with ice-cold PBS. After
centrifugation at 3,000 × g for 10 min, cells were resuspended
in ice-cold PBS containing protease inhibitors (Roche Applied Science, Castle
Hill, NSW, Australia) and sonicated briefly on ice. The supernatant was
collected after centrifugation at 1,000 × g for 10 min at 4
°C and protein estimation carried out using the Bio-Rad protein estimation
kit. Protein samples were analyzed on precast 4–20% iGel gradient gels
without SDS (Life Therapeutics, Frenchs Forest, NSW, Australia). Total cell
protein (20 μg) was mixed with 5× loading buffer (composition: 365
m m Tris-HCl, pH 6.8, 37.5% glycerol, 0.02% bromphenol blue, and 10%
SDS to yield SDS end concentrations of 2%) and denatured at 65 °C for 10
min. Samples were separated at 150 V for ~60 min and transferred to
nitrocellulose membrane for 1 h at 300 mA on ice in transfer buffer
(composition: 25 m m Tris, 192 m m glycine, 15% methanol,
pH 8.3). The nitrocellulose membrane was blocked overnight at 4 °C in
blocking buffer (composition: 130 m m NaCl, 2.7 m m KCl,
10 m m NaH 2PO 4, 1.8 m m
KH 2PO 4, 0.1% Tween-20, and 10% low-fat skim milk powder)
and incubated for 1 h at room temperature with rabbit anti-rat TRPV1 antibody
(1:5,000, Santa Cruz Biotechnology). A monoclonal anti-β-actin antibody
(1:20,000, clone AC-15, Sigma Aldrich) was also included for visualization of
β-actin to serve as a loading control. After washing in blocking buffer,
the membrane was incubated for 1 h with anti-rabbit and anti-mouse horseradish
peroxidase-conjugated secondary antibodies (both 1:5,000; Zymed Laboratories
Inc., Mount Waverley, Victoria, Australia). Blots were developed using ECL
Plus (GE Life Sciences, Rydalmere, NSW, Australia) for visualization of
chemiluminescence by exposure to ECL Hyperfilm (GE Life Sciences). The optical
density of bands was determined using MetaMorph Imaging Software (Version
6.2R5; Universal Imaging, Downington, PA). [3H]Resiniferatoxin Binding—For preparation of
total cell membranes to assess binding of [3H]resiniferatoxin to
the TRPV1, cells were incubated at 37 °C in a 5% humidified CO2
incubator with the appropriate treatment for 20–30 min, subsequently
placed on ice, and washed 2–3 times with ice-cold PBS. Cells were
dislodged with a cell scraper and collected by centrifugation at 3,000 ×
g for 5 min. The cells were resuspended in 1 ml of assay buffer
(composition: 5 mm KCl, 5.8 mm NaCl, 2 mm
MgCl2, 320 mm sucrose, 10 mm HEPES; pH 7.4)
and sonicated briefly. After centrifugation at 1,000 × g for 10
min, the supernatant was collected, and after protein estimation using a
Bio-Rad protein assay kit, diluted to 20 μg of protein/200 μl with assay
buffer containing an additional 0.25 mg/ml BSA. Plasma membrane samples for [ 3H]resiniferatoxin binding were
prepared according to the manufacturer's instructions with a Plasma Membrane
Protein Isolation kit (MBL International Corp, Woburn, MA) based on an aqueous
two-phase polymer system of dextran-polyethylene glycol, which isolates plasma
membrane proteins specifically with minimal contamination from other
intracellular membrane fractions
( 14,
15) (supplemental Fig. S1).
TRPV1/MOP HEK cells were incubated with the appropriate treatments in PSS at
37 °C for 20–30 min. Cells were placed on ice immediately, washed
with ice-cold PBS, and 5–10 × 10 7 cells collected by
centrifugation. The cell pellet was resuspended with 1 ml of the
homogenization buffer included in the kit and briefly sonicated. The resulting
homogenate was centrifuged at 700 × g for 10 min at 4 °C,
and the resulting supernatant collected for protein estimation. For each
treatment group, an identical amount of supernatant protein was utilized to
isolate purified plasma membrane fractions. The plasma membrane protein pellet
was collected by centrifugation at 44,800 × g for 30 min at 4
°C and resuspended in PBS for protein estimation. For
[ 3H]resiniferatoxin binding to the TRPV1, the plasma membrane
protein was diluted to 5 μg of plasma membrane protein/200 μl with assay
buffer containing 0.25 mg/ml BSA and used fresh. Quantification of TRPV1 binding was performed by homologous competitive
binding
( 16– 19)
in the presence of 50 p m [ 3H]resiniferatoxin
(PerkinElmer Life Sciences, 39.8 Ci/mmol) and varying concentrations of
unlabeled resiniferatoxin. Nonspecific binding was defined as occurring in the
presence of 1 μ m non-radioactive resiniferatoxin. For total cell
binding, 20 μg of protein was used, while for plasma membrane binding, 5
μg of purified plasma membrane fractions were sufficient. Using this
protocol, nonspecific binding was generally below 10–20%, as previously
reported ( 18). Binding
reactions were carried out by incubation at 37 °C for 1 h and terminated
by placing the assay mixtures on ice. Bound and free fractions were separated
with a cell harvester on Whatman GF/B filters that had been soaked in filter
buffer (composition: 50 m m Tris-HCl, 0.1% BSA, 0.5%
polyethyleneimine; pH 7.4) at 4 °C for at least 1 h. Samples were read on
a liquid scintillation counter after incubation of the filter papers with
~3 ml of liquid scintillant (OptiPhase HiSafe 3, PerkinElmer Life
Sciences) at room temperature for 10–14 h. The analysis of radioligand
binding data was performed using GraphPad Prism homologous competitive binding
analysis with one class of binding sites. Determination of Cell Viability by MTS Assay—The viability
of TRPV1/MOP HEK cells was determined using the CellTiter 96® Aqueous One
Solution Cell Proliferation Assay (Promega, Annandale, NSW, Australia)
according to the manufacturer's specifications. In brief, cells were grown to
~80–90% confluency on 96-well plates and treated with varying
concentrations of dansylcadaverine in PSS for 30 min. After a wash with PSS,
cells were incubated with the MTS reagent for another 2 h at 37 °C in 5%
CO2 and absorbance measured at 490 nm with a Bio-Rad model 550
microplate reader. Fluorescence Resonance Energy Transfer (FRET)
Measurements—FRET signals from murine homomultimeric TRPV1 channels
were determined as previously described
( 20). In brief, HEK293 cells
were transiently transfected with a C-terminal Cerulean fluorescence
protein-tagged TRPV1 construct (TRPV1-CFP) and a C-terminal enhanced yellow
fluorescence protein-tagged TRPV1 construct (TRPV1-YFP) using Lipofectamine
2000 (Invitrogen). Fluorescence imaging was carried out at room temperature
1–2 days after transfection using a fully automated inverted
fluorescence microscope (Olympus IX-81). For spectroscopic FRET measurements
from the plasma membrane, a spectrograph (Acton SpectraPro 2150i) was used in
conjunction with a Hamamatsu HQ CCD camera. Spectroscopic emission data
specifically from the plasma membrane or the cytoplasm was collected by
recording fluorescence intensity from the spectrograph slit location
corresponding to each cellular structure as previously described
( 21). FRET efficiency was
plotted as a function of the Cerulean to YFP fluorescence intensity ratio
(Fc/Fy). Best fit FRET models were fitted to the data as previously described
( 20) and were represented as
solid or dotted lines. Immunofluorescence—TRPV1/MOP HEK cells were plated on 25-mm
PDL-coated glass coverslips and grown to ~80–90% confluence. Cells
were treated for 30 min at 37 °C with the appropriate reagents in PSS and
placed immediately on ice to minimize receptor internalization. Cells were
washed twice in ice-cold PBS before a light fix with methanol/acetone (1:1) at
-20 °C for 30 min. After permeabilization with 0.2% Triton X in PBS for 10
min, cells were blocked by immersion in PBS with 3% BSA for 30 min. Rabbit
anti-rat TRPV1 primary antibody (1:100, Santa Cruz Biotechnology) was prepared
in PBS containing 3% BSA. After 1 h of incubation at room temperature, cells
were incubated with anti-rabbit fluorescein isothiocyanate-conjugated
secondary antibody (1:300) under low light for 1 h. Cells were mounted using
SlowFade AntiFade (Molecular Probes). Randomly selected images were viewed on
a Nikon Eclipse TE 300 inverted fluorescent microscope (excitation 488 nm,
emission 520 nm) and recorded with MetaFluor (Molecular Devices) imaging
software. Corel Photo-Paint (Corel, Ottawa, Ontario, Canada) was used for
processing of images. TRPV1 Expression in Calcium-rich Stores—As previously
described ( 22), to assess
TRPV1 expression in calcium-rich stores, the low affinity calcium probe
Fura-FF was sequestered into intracellular stores by loading TRPV1/MOP HEK
cells with Fura-FF-AM (12 μ m) for 2 h at 37 °C, followed by
washing with PSS for 15 min at 37 °C. Coverslips were transferred to the
recording chamber of an inverted Nikon Eclipse TE 300 fluorescent microscope
and viewed under a Nikon 40×/1.3 oil immersion objective lens.
Fluorescence signals from single cells were ratio-imaged by recording emission
intensity at 510 nm from excitation at 340 and 380 nm. Ratios of F340/F380
were depicted as pseudocolor images. Cells were then lightly fixed with 4%
paraformaldehyde for 20 min and stained for TRPV1 expression as described
above. Assessment of TRPV1 Multimers and Immunofluorescence in DRG from
Animals with Peripheral Inflammation—Ethical approval was obtained
from the University of Queensland Animal Ethics Committee and experiments
carried out in accordance with guidelines of the Committee for Research and
Ethical Issues of the International Association for the Study of Pain. Adult
male Wistar rats (250–300 g) were kept in a controlled environment at a
temperature of 22 ± 0.5 °C, relative humidity of 40–60%, and
a 12 h (6:30 AM to 6:30 PM) light-dark cycle with free access to standard lab
chow and tap water. To induce peripheral inflammation, 100 μl of Freund's
Complete Adjuvant (FCA, Sigma) was injected subcutaneously into the right hind
paw under light isoflurane anesthesia. L5 DRGs ipsilateral to the inflamed paw
were isolated 6–7 days after induction of inflammation. For analysis of
TRPV1 multimerization in DRG neurons, individual L5 DRG were placed
immediately in ice-cold PBS containing protease inhibitors (Roche Applied
Sciences), minced with surgical scissors and sonicated on ice. Protein samples
were collected after centrifugation at 1,000 × g for 10 min and
20 μg of protein analyzed by SDS-PAGE and Western blotting as described
above. For immunofluorescence studies of plasma membrane TRPV1 expression, L5 DRG
contralateral and ipsilateral to the inflamed paw were harvested and
immediately fixed for 2–3 h in ice-cold paraformaldehyde (4%). DRGs were
then transferred to 0.32 m sucrose and cryoprotected at 4 °C
for at least 24 h. Tissues were embedded in Tissue-Tek® O.C.T. Compound
(Sakura Finetek, Torrance, CA), cut to 10-μm cryostat sections, and mounted
on SuperFrost Ultra Plus® tissue adhesion slides (Menzel GmbH Co KG,
Braunschweig, Germany) before immunofluorescence labeling as described
above. Data Analysis—Unless otherwise states, all graphs are
representative of at least two to three independent experiments with minimum
n values for each treatment group being n = 3. Unless
individual data points are shown, data are presented as means ± S.E. of
the mean (S.E.). As mentioned above, statistical analysis and fitting of
4-parameter Hill equations were carried out using GraphPad Prism Version 4.03.
Statistical significance was defined as p < 0.05 and determined
using an unpaired, two-tailed Student's t test where appropriate.
Calcium traces were analyzed by statistical comparison of maximum ΔF/F
values using an unpaired, two-tailed Student's t test with
statistical significance defined as p < 0.05. Capsaicin Responses Potentiated by cAMP-dependent PKA Are Inhibited by
Morphine—Potentiation of TRPV1-mediated Ca 2+ responses
to addition of varying concentrations of capsaicin was assessed after a short
(15 min) incubation with 0.1% DMSO (control) or forskolin (50
μ m, ). Treatment with the adenylate cyclase activator
forskolin leads to an increase in cellular cAMP levels and resultant
activation of cAMP-dependent PKA. PKA-mediated phosphorylation of TRPV1 in
turn causes sensitization of TRPV1 responses
( 23). While cAMP analogues
acting directly at PKA lead to a similar sensitization, opioids, by virtue of
their antihyperalgesic activity resulting from adenylate cyclase inhibition,
are unable to prevent TRPV1 sensitization by direct PKA activators
( 12). Thus, to assess the
opioid-sensitive mechanisms involved in TRPV1 sensitization by cAMP-dependent
PKA, forskolin was used to potentiate TRPV1 responses. Consistent with
previously described studies
( 12), treatment with forskolin
significantly increased TRPV1-mediated capsaicin responses
(, p < 0.001). The (MOP) agonist morphine
inhibited this potentiation () through inhibition of forskolin-stimulated cAMP
production, which was maximal at 1 μ m morphine, although
reversal of forskolin-stimulated cAMP accumulation by morphine was not
complete (,
inset). We then focused on assessing the mechanism of potentiation of
TRPV1 by forskolin. Treatment of TRPV1/MOP HEK Cells with the PKA Activator Forskolin
Increases TRPV1 Multimerization—Augmentation of TRPV1 responses by
forskolin ()
appears consistent with an increase in functional TRPV1 channels. However, it
is unclear how forskolin could achieve an increase in functional TRPV1
channels in a way that would be consistent with rapid and dynamic regulation
of nociceptive TRPV1 signaling. As functional TRPV1 channels are proposed to
exist in homomultimeric form
( 14,
19– 23),
one mechanism for cAMP-mediated promotion of TRPV1 responses is assembly of
pre-existing plasma membrane TRPV1 monomers to form new functional plasma
membrane multimeric TRPV1 channels. We assessed total cell TRPV1 multimerization by SDS-PAGE under non-reducing
conditions, as described previously
( 13,
24). TRPV1 monomers and
multimers are detectable by SDS-PAGE under non-reducing conditions
( 13). Indeed, we observed
bands consistent with glycosylated and unglycosylated TRPV1 monomers (97 and
113 kDa, respectively) as well as a multimeric TRPV1 species (~200 kDa)
under these conditions (). This multimeric species represents a TRPV1 dimer,
which is particularly stable, has previously been demonstrated to parallel
formation of the less stable tetramer, and can be used to assess
oligomerization of TRPV1 ( 13,
25). TRPV1/MOP HEK cells were treated with 0.1% DMSO (control), morphine (1
μm), or forskolin (500 μm, FSK) containing in
addition morphine (FSK+morphine) as appropriate
(). Treatment with forskolin significantly (p
< 0.05 by ANOVA) increased TRPV1 multimer formation, while pretreatment
with morphine reversed this increase (). Treatment with the Transglutaminase Inhibitor Dansylcadaverine Inhibits
Capsaicin Responses Potentiated by Forskolin—To determine if TRPV1
functionality is affected when TRPV1 multimers are disrupted, we preincubated
TRPV1/MOP HEK cells with varying concentrations of the transglutaminase
inhibitor dansylcadaverine and assessed calcium responses to the addition of
300 n m capsaicin in control and forskolin-stimulated cells
().
Preincubation with dansylcadaverine at previously reported concentrations
( 26) inhibited capsaicin
responses in control and forskolin-treated cells, consistent with the
functional form of the TRPV1 as a multimer
( 14,
20– 23).
Forskolin-treated cells were more sensitive to dansylcadaverine (log EC 50
(Control) -3.185 ± 0.027; log EC 50 FSK -3.269 ±
0.013; p < 0.05). The inhibition of capsaicin responses by
dansylcadaverine was not due to impaired cell viability, globally impaired
calcium responses, or TRPV1 antagonism
().
There was no displacement of the radiolabeled TRPV1 agonist
[ 3H]resiniferatoxin by unlabeled dansylcadaverine (100
μ m to 1.2 m m), indicating that the transglutaminase
inhibitor does not act as a competitive TRPV1 antagonist
().
Preincubation with dansylcadaverine did not affect responses to the calcium
ionophore ionomycin (10 μ m,
), or cell
viability over this period (). Inhibition of forskolin-stimulated capsaicin
responses by dansylcadaverine appears to be a consequence of inhibition of
functional TRPV1 multimer formation. Forskolin Does Not Increase Multimerization of TRPV1 Monomers in the
Plasma Membrane—Last, to assess the effect of forskolin on changes
in the proportion of plasma membrane TRPV1 multimers and monomers, we
quantified TRPV1 multimerization utilizing a well characterized spectroscopy
FRET model ( 20) that allows
quantification of FRET signals specifically from the plasma membrane. HEK
cells were transiently transfected with Cerulean fluorescence protein-tagged
TRPV1 (TRPV1-CFP) and enhanced yellow fluorescence protein-tagged TRPV1
(TRPV1-YFP) and the FRET signal determined as previously described
( 20) prior to and after
treatment with forskolin (500 μ m). No increase in FRET signal, a
measure of de novo formation of TRPV1 multimers from pre-existing
plasma membrane monomers, was detected
(). Hence, increases in
cellular cAMP do not cause multimerization of TRPV1 monomers in the plasma
membrane; rather, our results are consistent with a model whereby forskolin
increases plasmalemmal TRPV1 multimers from an internal pool of monomers. TRPV1 Multimers Are Mainly Localized to the Plasma
Membrane—To determine if the intracellular pool of TRPV1 available
for plasma membrane insertion was indeed in a multimeric or monomeric state,
we compared the relative proportion of TRPV1 multimers and monomers in
intracellular pools and the plasma membrane. If forskolin was to promote the
assembly and rapid translocation of newly formed TRPV1 multimers to the plasma
membrane from a pool of intracellular TRPV1 monomers, the majority of TRPV1
monomers would be found intracellularly, with a correspondingly large
proportion of TRPV1 multimers existing on the plasma membrane. Assessment of
the FRET signal from the plasma membrane and intracellular compartments
clearly demonstrated that TRPV1 multimers occur mainly on the plasma membrane,
while the correspondingly lower FRET signal from intracellular compartments is
consistent with a higher proportion of TRPV1 monomers
(). To assess
the functionality of TRPV1 in these internal pools, we determined the presence
of TRPV1 on calcium-rich intracellular compartments and monitored calcium
release from stores in response to capsaicin. The low affinity ratiometric
calcium dye Fura-FF-AM (12 μm) was sequestered into
intracellular stores. High Fura-FF fluorescence ratios (F340/F380) indicative
of high calcium levels were found in similar patterns to TRPV1
immunofluorescence (). Although the TRPV1 was clearly expressed in
calcium-rich intracellular compartments, addition of capsaicin did not
increase cytosolic free calcium levels in the absence of extracellular calcium
(). This is
consistent with an intracellular pool of inactive monomeric TRPV1 being
available for plasma membrane insertion, where it exists in a multimeric
state. Augmentation of TRPV1 Activity Is Associated with TRPV1 Translocation
to the Plasma Membrane—Translocation of TRPV1 channels to the
plasma membrane was assessed in TRPV1/MOP HEK cells treated with 0.1% DMSO
(control), forskolin (500 μm, FSK), or morphine with forskolin
(1 μm, FSK+morphine). As predicted by our model, treatment with
forskolin caused a marked translocation of TRPV1 immunoreactivity to the
plasma membrane (), resulting in distinct membrane labeling.
Pretreatment with morphine inhibited the predominant plasma membrane TRPV1
expression pattern and reversed TRPV1 labeling to mixed plasma membrane and
cytoplasmic localization (). To exclude the possibility that treatment with
forskolin could increase de novo synthesis of TRPV1 channels on the
plasma membrane, we further assessed TRPV1 binding density in isolated plasma
membrane protein fractions and total cell protein
() using homologous competitive binding analysis. Plasma membrane TRPV1 radioligand binding density in TRPV1/MOP HEK cells
treated with forskolin (500 μm, FSK) was significantly increased
(p < 0.05, Bmax FSK, 1284.6 ± 419.2 pmol/mg
protein) compared with control (, Bmax control, 685.1 ± 310 pmol/mg
protein). In agreement with our immunofluorescence studies, pretreatment with
morphine reversed this increased plasma membrane binding density
(Bmax FSK+morphine, 331.3 ± 156.1 pmol/mg protein;
). Consistent
with our model, total cell [3H]resiniferatoxin binding was
unaffected by treatment with forskolin or morphine
(, p
> 0.05), indicating that the increase in plasma membrane TRPV1 expression
was due to translocation of TRPV1 channels rather than increased formation of
new TRPV1 protein. TRPV1 Multimers and Plasma Membrane Expression in
Inflammation—We assessed whether two pools of TRPV1 (multimers and
monomers) are present in vivo during inflammation, a process where
signaling through cAMP-dependent PKA is increased
( 27). Western blot analysis
demonstrated that, as previously suggested, TRPV1 protein was present in
inflamed DRG in two distinct pools representing monomeric and multimeric
species (). The
presence of monomeric and multimeric TRPV1 provides the basis for rapid
dynamic regulation of TRPV1 responses via the mechanisms we propose in
addition to increased TRPV1 expression in inflammation
( 28– 31).
Contribution of these pathways to in vivo TRPV1 sensitization is also
suggested by the increased TRPV1 plasma membrane staining patterns in L5 DRG
ipsilateral to the inflamed hind paw (). Potentiation of TRPV1 responses by cAMP-dependent PKA is crucial to the
development of inflammatory hyperalgesia. Although PKA consensus sites for
TRPV1 have been identified
( 32,
33), the molecular pathways
leading to the rapid sensitization of the receptor as a result of PKA-mediated
phosphorylation are not fully understood. Our results propose a new mechanism
describing how TRPV1 potentiation by cAMP-dependent PKA can be achieved
rapidly without the need for translational changes, while maintaining the
capacity for dynamic modulation of sensitized TRPV1 signaling. Our results
propose a mechanism for potentiation of TRPV1 responses by cAMP-dependent PKA
incorporating both increased TRPV1 translocation and multimerization
(). The dynamic
regulation of these pathways by opioids represents a cellular mechanism
allowing for rapid modification of the hyperalgesic or sensitized component of
TRPV1 signaling. The TRPV1 is a target for inflammatory signaling through cAMP-dependent
PKA. As an activator of adenylate cyclase causing elevation of cellular cAMP
levels, forskolin affects potentiation of TRPV1 responses as a result of
activation of cAMP-dependent PKA
( 2,
23). Potentiation of
TRPV1-mediated capsaicin responses by forskolin is consistent with an increase
in functional TRPV1 channels and indeed, increased TRPV1 expression occurs in
inflammation ( 29,
31). However, as the
potentiation of capsaicin responses by forskolin occurs rapidly, pathways
other than increased transcription of TRPV1 must be involved and were assessed
in this study. The functional TRPV1 channel exists most likely as homomultimers, whereby
monomeric TRPV1 subunits assemble in tetrameric stoichiometry around the
aqueous pore ( 13,
34). The most compelling
confirmation of the functional importance of correct assembly of TRPV1
subunits arguably arises from the observation that incorporation of a single
dominant negative mutant into the TRPV1 channel complex completely abolishes
capsaicin responses ( 34).
Surprisingly, despite clear evidence that TRPV1 multimerization is essential
for channel function, regulation of multimerization has not been described as
a possible contributor to post-translational modification of TRPV1 function.
An increase in TRPV1 multimerization would provide a cellular mechanism for
achieving rapid modulation of TRPV1 signaling without the need for altered
transcription. Indeed, we found that treatment with forskolin significantly
increased TRPV1 multimers in total cell lysates, indicating that the rapid
potentiation of TRPV1 responses after treatment with forskolin might involve
the generation of new functional units of TRPV1. Pretreatment with morphine
prevented a significant increase in TRPV1 multimerization, providing evidence
that this system could present a pathway for rapid and dynamic modulation of
TRPV1 potentiation. Activation of adenylate cyclase can increase transglutaminase activity and
expression ( 32,
33), and TRPV1 cross-linking
has previously been demonstrated to involve transglutaminases
( 13). Thus, we assessed the
ability of forskolin to potentiate TRPV1-mediated capsaicin responses under
conditions where TRPV1 multimers are disrupted. In accordance with increased
TRPV1 multimerization in response to forskolin, capsaicin responses were
sensitive to the transglutaminase inhibitor dansylcadaverine, demonstrating
the importance of functional TRPV1 multimers in mediating TRPV1 potentiation
by forskolin. To assess the effect of forskolin on changes in the proportion of plasma
membrane TRPV1 multimers and monomers, we quantified TRPV1 multimerization by
FRET. Increased multimerization of TRPV1 monomers in a mixed population of
plasma membrane TRPV1 subunits would result in an increase in FRET. The FRET
signal from the plasma membrane was not affected by forskolin, indicating that
the increase in TRPV1 multimers did not result from an increase in
cross-linking of pre-existing monomers in the plasma membrane. As FRET is not
affected by absolute fluorescence intensity and thus insensitive to insertion
of preformed multimers into the plasma membrane, this would suggest that
rather than promoting multimerization of TRPV1 monomers in the plasma
membrane, forskolin might affect insertion of TRPV1 multimers into the plasma
membrane. However, simple translocation of functional TRPV1 multimers
preformed in intracellular stores would not only be inconsistent with an
overall increase in TRPV1 multimerization, but in addition would likely lead
to major alteration of calcium homeostasis in intracellular stores. An
intracellular store of inactive TRPV1 monomers, however, would provide a
source of TRPV1 subunits available for translocation to the plasma membrane
that would not produce major disruptions in calcium homeostasis. These
monomers are likely to be translocated to the plasma membrane, where they
become inserted as functional TRPV1 multimers, thus providing a physiological
mechanism for rapid assembly of functional TRPV1 units. Consistent with this
model, the plasma membrane would be expected to contain significantly more
TRPV1 multimers than the intracellular space. This was indeed shown to be the
case, as evaluation of the FRET signals from intracellular and plasma membrane
regions revealed that TRPV1 multimers are more abundant in the plasma membrane
than in intracellular regions. Further functional evidence supporting our proposed model stems from the
observation that TRPV1 does not mediate significant calcium release from
intracellular stores at concentrations that cause considerable influx of
calcium through the plasma membrane. Several studies report TRPV1 expression
in intracellular structures such as the endoplasmic reticulum, both in native
as well as overexpressed cell systems
( 31– 33),
and TRPV1 expression in calcium-rich intracellular stores was confirmed here.
If intracellular TRPV1 channels were indeed pre-assembled as functional
channels, this should translate into effective release of calcium from
intracellular stores upon channel activation, as TRPV1 agonists such as
capsaicin are highly lipophilic and readily cross the cell membrane
( 35). While some studies have
attributed calcium release channel properties to the TRPV1, calcium release
from stores generally occurred at significantly higher capsaicin
concentrations than those required to elicit calcium influx through plasma
membrane TRPV1
( 31– 33).
Indeed, EC 50 values of up to 13 μ m are reported for
capsaicin-mediated calcium release from intracellular stores
( 36), while the corresponding
EC 50 for calcium influx through plasma membrane TRPV1 is
~30–40 n m
( 12,
37). Because high capsaicin
concentrations are associated with nonspecific effects including activation of
phospholipase C ( 38),
mechanisms other than TRPV1 activation likely contribute to the release of
calcium from intracellular stores in response to high concentrations of
capsaicin. Similarly, the TRPM8 agonist menthol appears to release calcium
from the ER and Golgi via a TRPM8-independent mechanism
( 39). We systematically
compared capsaicin dose response curves in the presence and absence of
extracellular calcium and found that, consistent with a largely inactive pool
of TRPV1 in intracellular structures, negligible store release occurs in
response to capsaicin concentrations that cause significant calcium influx
through the plasma membrane. Indeed, an intracellular reservoir of active
TRPV1 channels could be detrimental to cellular calcium homeostasis given that
TRPV1 agonists are generally lipophilic
( 35). Promotion of TRPV1 multimerization from intracellular monomers by forskolin
would require rapid translocation of these TRPV1 channels to the plasma
membrane. Consistent with studies describing mixed intracellular and plasma
membrane localization for TRPV1 in both native cells as well as overexpressed
systems ( 22,
31,
32), control cells displayed
significant expression of TRPV1 in intracellular structures, while some
punctuate TRPV1 localization was apparent in the plasma membrane. In contrast,
treatment with forskolin resulted in a striking shift in TRPV1
immunofluorescence to the plasma membrane, resulting in a distinct ring-like
pattern. Pretreatment with morphine demonstrated that this translocation can
be dynamically modulated, as the predominant plasma membrane localization of
TRPV1 was largely lost. Increased TRPV1 translocation by forskolin and
inhibition thereof by morphine was further confirmed by quantification of
[ 3H]resiniferatoxin binding to purified plasma membrane fractions.
These findings demonstrate that the mechanisms underlying rapid potentiation
of TRPV1-mediated capsaicin responses by the adenylate cyclase activator
forskolin can be dynamically modulated by morphine, an observation that could
be of particular relevance to inflammatory hyperalgesia. Such opioid-mediated
modulation of TRPV1 multimerization and trafficking could, in addition to
effects of opioids on other inflammatory mediators
( 40– 46),
also contribute to the enhanced anti-nociceptive effect of opioids in
inflammation. The exact pathways involved in trafficking and multimerization of TRPV1 by
cAMP-dependent PKA remain to be elucidated. Notably, both PKA phosphorylation
sites as well as putative SNARE interaction sites are located on the
N-terminal of TRPV1 ( 8,
47,
48), such that phosphorylation
of TRPV1 by PKA could lead to enhancement of these exocytosis pathways through
to-date unknown mechanisms. Indeed, PKA-dependent trafficking pathways have
been reported for a number of receptors and ion channels including the
chloride-selective anion channel CFTR and AMPA receptor
( 49,
50). Alternatively, increased
multimerization and translocation of TRPV1 could occur as a downstream result
of PKA-mediated phosphorylation of other effectors rather than as a direct
result of phosphorylation of TRPV1 by PKA. Indeed, TRPV1 has been reported to
bind to several proteins involved in receptor trafficking, including Snapin
and synaptotagmin IX as well as eferin
( 48,
51). The function of these
proteins in turn can be affected by PKA-mediated phosphorylation
( 52). As several other
proteins have been reported to regulate trafficking of various trp channels
( 53), the exact pathways
involved in PKA-mediated TRPV1 trafficking remain to be identified. Similarly, in addition to increased transglutaminase activity and
expression as a result of activation of adenylate cyclase
( 32,
33), other pathways not
implicated to date may be involved in increased multimerization of TRPV1 by
cAMP-dependent PKA. Speculatively, TRPV1 multimerization might occur in lipid
rafts in the plasma membrane due to the presence of to-date unidentified
accessory proteins. Indeed, depletion of cholesterol can inhibit TRPV1
function and plasma membrane expression
( 54), supporting the notion
that correct TRPV1 assembly to functional multimers occurs in discreet areas
of the plasma membrane. Once the exact pathways involved are identified, these
would present themselves as therapeutic targets to modulate TRPV1-mediated
hyperalgesia. In conclusion, our findings point toward a mechanism that allows for rapid
and dynamic modulation of TRPV1 responses. Insertion of functional TRPV1
multimers into the plasma membrane from an intracellular store of inactive
monomers as a consequence of activation of cAMP-dependent PKA allows for rapid
potentiation and modulation of TRPV1 responses and conveys the additional
advantage of minimizing disruption of intracellular calcium stores. This model
further extends the complexity of cellular regulation of TRPV1 potentiation
beyond the simple increase in trafficking that is suggested to mediate TRPV1
sensitization by protein kinase C
( 37– 39).
In addition, this system provides the capacity for rapid potentiation of TRPV1
responses without having to rely on transcriptional changes. Modification of
the cellular mechanisms that lead to multimerization of TRPV1 channels and
insertion of functional channels into the plasma membrane provides an
endogenous pathway for adjusting hyperalgesia, without altering nociception
itself. The propensity for dynamic regulation of potentiated TRPV1 responses
is demonstrated by the ability of morphine to inhibit forskolin-mediated
potentiation of TRPV1 responses by modulating TRPV1 multimerization and plasma
membrane trafficking. This model may be of particular relevance to
inflammation, where cellular cAMP levels are elevated, and the resultant
increased activity of cAMP-dependent PKA contributes to the development of
hyperalgesia ( 7,
27), but may also apply to
other potentiation pathways. We thank Dr. Alpha Yap (Institute for Molecular Biosciences, The University
of Queensland, St Lucia, Australia) for the gift of the rabbit E-cadherin
antibody. Part of the work was conducted in a UC Davis facility constructed
with support from Research Facilities Improvement Program Grant
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