Calcium-dependent desensitization of vanilloid receptor TRPV1: a mechanism possibly involved in analgesia induced by topical application of capsaicin.

Abstract

The rationale for the topical application of capsaicin and other vanilloids in the treatment of pain is that such compounds selectively excite and subsequently desensitize nociceptive neurons. This desensitization is triggered by the activation of vanilloid receptors (TRPV1), which leads to an elevation in intracellular free Ca2+ levels. Depending on the vanilloid concentration and duration of exposure, the Ca2+ influx via TRPV1 desensitizes the channels themselves, which may represent not only a feedback mechanism protecting the cell from toxic Ca2+ overload, but also likely contributes to the analgesic effects of capsaicin. This review summarizes the current state of knowledge concerning the mechanisms that underlie the acute capsaicin-induced Ca2+-dependent desensitization of TRPV1 channels and explores to what extent they may contribute to capsaicin-induced analgesia. In view of the polymodal nature of TRPV1, we illustrate how the channels behave in their desensitized state when activated by other stimuli such as noxious heat or depolarizing voltages. We also show that the desensitized channel can be strongly reactivated by capsaicin at concentrations higher than those previously used to desensitize it. We provide a possible explanation for a high incidence of adverse effects of topical capsaicin and point to a need for more accurate clinical criteria for employing it as a reliable remedy.

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PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)
© 2008 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres

Physiol. Res. 57 (Suppl. 3): S59-S68, 2008

MINIREVIEW

Calcium-Dependent Desensitization of Vanilloid Receptor TRPV1:
A Mechanism Possibly Involved in Analgesia Induced by Topical
Application of Capsaicin


L. VYKLICKÝ, K. NOVÁKOVÁ-TOUŠOVÁ, J. BENEDIKT, A. SAMAD, F. TOUŠKA,
V. VLACHOVÁ

Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the
Czech Republic, v.v.i , Prague, Czech Republic

Received February 15, 2008
Accepted April 16, 2008
On-line May 13, 2008


Summary
The rationale for the topical application of capsaicin and other
vanilloids in the treatment of pain is that such compounds
selectively excite and subsequently desensitize nociceptive
neurons. This desensitization is triggered by the activation of
vanilloid receptors (TRPV1), which leads to an elevation in
intracellular free Ca2+ levels. Depending on the vanilloid
concentration and duration of exposure, the Ca2+ influx via
TRPV1 desensitizes the channels themselves, which may
represent not only a feedback mechanism protecting the cell
from toxic Ca2+ overload, but also likely contributes to the
analgesic effects of capsaicin. This review summarizes the
current state of knowledge concerning the mechanisms that
underlie the acute capsaicin-induced Ca2+-dependent
desensitization of TRPV1 channels and explores to what extent
they may contribute to capsaicin-induced analgesia. In view of
the polymodal nature of TRPV1, we illustrate how the channels
behave in their desensitized state when activated by other stimuli
such as noxious heat or depolarizing voltages. We also show that
the desensitized channel can be strongly reactivated by capsaicin
at concentrations higher than those previously used to
desensitize it. We provide a possible explanation for a high
incidence of adverse effects of topical capsaicin and point to a
need for more accurate clinical criteria for employing it as a
reliable remedy.

Key words
TRPV1 receptors
• Capsaicin
• Topical application
•
Desensitization • Analgesia
Corresponding author
V. Vlachová, Institute of Physiology AS CR, Vídeňská 1083, 142
20 Prague 4, Czech Republic, Fax. 420-29644-2488, E-mail:
vlachova@biomed.cas.cz

Introduction

Capsaicin produces burning pain in humans when
brought into contact with the corium or mucosa and,
paradoxically, its topical application to the skin has been
found useful in the treatment of some pain states in
traditional and contemporary medicine (for reviews, see
Szallasi and Blumberg 1999, Sawynok 2003, Nagy et al.
2004). Experimental evidence has been presented that
capsaicin causes persistent functional desensitization of
polymodal primary nociceptors after repeated or prolonged
application (Jancso et al. 1967, Carpenter and Lynn 1981,
Szolcsanyi 2004). This desensitization was suggested to
occur rather due to physiological than to morphological
alterations (McMahon et al. 1991), however, later
convincing evidence was presented that repeated capsaicin
application to the skin produces a degeneration of
epidermal nerve fibers that may underlie this type of
analgesia (Simone et al. 1998, Nolano et al. 1999).
Although capsaicin and other vanilloids have been shown
to disrupt some vital organelles and promote apoptosis in
various cell types (Hail 2003 and references therein), most
of the well characterized effects are mediated through the
activation of a specific receptor-ion channel that is natively

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expressed in mammalian primary nociceptors (Caterina et
al. 1997). This receptor was termed vanilloid receptor
subtype 1 (TRPV1) because it became the founding
member of the vanilloid receptor subfamily of transient
receptor potential (TRP) channels, a large superfamily of
nonselective cation channels that play an important role in
many sensory functions (for recent reviews see Clapham
2003, Planells-Cases et al. 2005, Tominaga and Tominaga
2005, Bandell et al. 2007, Nilius et al. 2007, Pingle et al.
2007, Venkatachalam and Montell 2007). Although the
molecular identification of the TRPV1 channels has greatly
facilitated understanding the mechanisms by which
vanilloids increase and subsequently attenuate nociceptor
activity, there is still no general consensus about the in vivo
role of these mechanisms in the development of vanilloid-
induced analgesia. What is clear from research carried out
to date is that this process is triggered by Ca2+ influx via
TRPV1, which leads to desensitization of the channel
protein complex itself and, in extreme cases, to the
degeneration of TRPV1-expressing sensory neurons due to
Ca2+ overload (Olah et al. 2001, Szolcsanyi 2004, Tender
et al. 2005).
In vitro studies on native and recombinant
channels demonstrate that TRPV1 exhibits two types of
desensitization, both of which frequently occur in
conjunction (Figs 1A and 1C): acute desensitization,
which is a diminished response during a continuous
vanilloid application, and tachyphylaxis, which is a


Fig. 1. Effects of extracellular Ca2+ on acute desensitization of capsaicin-activated currents in HEK293T cells transfected with rat TRPV1.
(A) Whole-cell current responses to consecutive applications of 1 µM capsaicin induced in a nominally Ca2+-free extracellular solution (a
and b) and in standard bath solution containing 1 mM Ca2+ (c and d). The methods used in this study are described in detail in
Novakova-Tousova et al. (2007). Briefly, human embryonic kidney (HEK) 293T cells, transiently transfected with recombinant plasmid
cDNA encoding wild-type rat TRPV1, were used for whole-cell recordings using a conventional patch-clamp technique. The cells were
superfused with an extracellular control solution (ECS) (mM): NaCl, 160; KCl, 2.5; CaCl2, 1; MgCl2, 2; HEPES, 10; glucose 10. The
intracellular pipette solution contained (mM): Cs-gluconate, 125; CsCl, 15; EGTA, 5; HEPES, 10; CaCl2, 0.5; MgATP, 2; pH was adjusted
to 7.3 with CsOH. Holding potential was -70 mV. Capsaicin was applied for the durations indicated by the bars above the records.
Dashed lines indicate zero current level. (B) Barium cannot substitute for Ca2+ in acute capsaicin-induced desensitization of the TRPV1
channel. Capsaicin (1 µM) was applied either in Ca2+-free solution to which 1 mM EGTA was added (a, c) or in the bath solution in
which Ba2+ (2 mM) was substituted for Ca2+ (b). (C) Whole-cell membrane currents from another TRPV1-HEK293T cell. Capsaicin was
applied in Ca2+-free solution to which 1 mM EGTA was added to chelate residual Ca2+ (0 Ca2+) or in solution containing 2 mM Ca2+.

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Acute Desensitization of TRPV1 S61

reduction in the response to repeated applications
(Docherty et al. 1996, Koplas et al. 1997, Bhave et al.
2003). Over the past decade, it has become apparent that
a predominant fast (acute) component of TRPV1
desensitization, in both native and overexpression
contexts, critically depends on Ca2+ influx through the
channel (Koplas et al. 1997, Piper et al. 1999, Mohapatra
and Nau 2003, Numazaki et al. 2003, Rosenbaum et al.
2004, Lishko et al. 2007). Although it is not yet certain
whether the acute desensitization and tachyphylaxis share
the same cellular mechanisms (Docherty et al. 1996,
Koplas et al. 1997), there is good evidence that it is the
acute phase that accounts for most of the diminution of
responsiveness occurring within the first few seconds
after vanilloids are applied for the first time to a cell
(Cholewinski et al. 1993, Liu and Simon 1996).
In this review, we present an update on findings
concerning the mechanisms that underlie acute capsaicin-
induced Ca2+-dependent desensitization of the TRPV1
channel and argue that further study is needed to clarify
the contribution of these mechanisms to capsaicin-
induced analgesia.

No single molecular mechanism alone (such
as the dephosphorylation of TRPV1) can
account for the whole phenotype of
capsaicin-induced Ca2+-dependent desensiti-
zation

Among the Ca2+-activated enzymes that were
reported to modulate TRPV1 desensitization is the
Ca2+/calmodulin-dependent serine/threonine phosphatase
2B, calcineurin, which
receptors (Docherty et al. 1996, Mohapatra and Nau
2005). Conversely, phosphorylations at several consensus
sites for protein kinase C (PKC), cAMP-dependent
protein kinase A (PKA) and Ca2+/calmodulin dependent
kinase II (CaMKII) have been shown to reduce the Ca2+-
mediated desensitization of TRPV1. There are a number
of putative phosphorylation sites at which the TRPV1
channel can be regulated: S502 and S800 have been
implicated as targets of PKC-dependent phosphorylation
(Vellani et al. 2001, Numazaki et al. 2002, Bhave et al.
2003, Mandadi et al. 2004, Mandadi et al. 2006) and
S116, T144, T370, and S502 were identified as the key
sites at which PKA phosphorylation increases the open
probability and reverses desensitization of the channel
(Bhave et al. 2002, Mohapatra and Nau 2003, Mohapatra
and Nau 2005). A coincident phosphorylation of S502


dephosphorylates TRPV1
and T704 by CaMKII has been proposed to be a
necessary condition for the vanilloid binding capacity of
TRPV1 (Jung et al. 2004). From these findings it was
inferred that it is mostly a functional reflection of the
dynamic balance between
phosphorylation and dephosphorylation of the receptor
protein that accounts for the desensitization of the
TRPV1 channel. Later, evidence was presented that the
TRPV1 desensitization might involve a much more
complex Ca2+-dependent
dependent TRPV1 desensitization has been shown to be
accompanied by a profound change in voltage
dependence (Piper et al. 1999, Gunthorpe et al. 2000),
loss of capsaicin binding (Jung et al. 2004), and depletion
of membrane phosphatidylinositol 4,5-bisphosphate
(PIP2), indicative of the involvement of Ca2+-dependent
PLC activity in this process. A role for ATP was also
suggested, based on the evidence that the recovery from
desensitization requires a high cytoplasmic concentration
of ATP to replenish PIP2 (Liu et al. 2005). Mutation of
the aromatic tyrosine residue Y671 within the internal
pore of the TRPV1 channel greatly reduced Ca2+-
dependent desensitization, suggesting that this residue is
also involved in the structural rearrangements of the
channel protein complex (Mohapatra et al. 2003).
Although in many cases it could not be clearly
distinguished which of these processes are primarily
direct causes of desensitization rather than consequences
of TRPV1 activation, it became clear that the
dephosphorylation of TRPV1 represents only one of the
possible mechanisms involved in analgesia after the
topical application of capsaicin.

The prevention of acute desensitization of the
TRPV1 channel by substituting Ba2+ for Ca2+
highlights a possible role for calmodulin

Structurally, like other members of the TRP
channel superfamily, TRPV1 channels are homotetramers
assembled around a centrally located aqueous pore (for a
review, see Owsianik et al. 2006). Each subunit contains
six transmembrane spanning domains (S1-S6) with a
pore-lining P region linking the S5 and S6 domains.
Although the crystal structure of the transmembrane part
is not yet available for any of the TRP channels, it is
generally accepted that it is analogous to the molecular
architecture of the voltage-gated potassium channels: S1-
S4 are putatively located on or close to the protein-lipid
interface, whereas the four S5-P-S6 segments form the
the Ca2+-dependent
pathways. Acute Ca2+-

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pore. Upon activation, Ca2+ permeates the TRPV1
channel pore more readily than other external cations
(PCa/PNa ~ 10, PMg/PNa ~ 5). So, at negative membrane
potentials, the driving force is favorable for a strong Ca2+
influx. If Ba2+ is used as the charge carrier instead of Ca2+
in whole-cell patch-clamp experiments on cells that
express TRPV1 channels, capsaicin induces responses
that do not desensitize and only slightly decrease in
magnitude when compared to the responses induced by
capsaicin in Ca2+-free extracellular solution (Fig. 1B).
Although consistent effects are observed in heterologous
expression systems (Gunthorpe et al. 2000, McNamara et
al. 2005), earlier studies found that Ba2+ supports acute
desensitization and tachyphylaxis of capsaicin-induced
responses in native TRPV1 channels in DRG neurons
isolated from rats, a result which has been interpreted by
Koplas et al. (1997) as showing that Ba2+ can activate
calcineurin-dependent dephosphorylation independently
of calmodulin. This important calcium downstream
effector was proposed to mediate TRPV1 desensitization
more than a decade ago (Docherty et al. 1996) and this
role has been debated ever since. Ba2+ as a surrogate for
Ca2+ substitutes poorly at Ca2+-sensitive regulatory sites
and is also a poor substrate for Ca2+ transport
mechanisms. Apparently because of its larger effective
ionic radius than Ca2+ (1.38 Å versus 1.06 Å), Ba2+ also
does not bind to calmodulin (CaM) (Chao et al. 1984).
More recently, the TRPV1 channel has been shown to
directly bind CaM at two sites in both a Ca2+-dependent
and Ca2+-independent manner. One of these sites was
localized in the N-terminal region (Rosenbaum and
Gordon 2002, Lishko et al. 2007) whereas the other has
been identified in the C-terminal domain (Numazaki et al.
2003). One of the most comprehensive models for the
Ca2+-induced desensitization of the TRPV1 channel has
emerged from these studies (Fig. 2). The picture is built
largely on recent evidence suggesting that the cytosolic
ankyrin-repeat domain of TRPV1 (residues 101-364)
binds ATP and that calmodulin, in a Ca2+-dependent
manner, competes with ATP at the same interaction site
(Lishko et al. 2007). These two competitors have
opposite effects on TRPV1 channel activity: ATP
sensitizes the channel whereas Ca2+-CaM inhibits it.

The apparent affinity for capsaicin is
decreased in a desensitized TRPV1 channel

Acute TRPV1 desensitization depends not only
on the amount of calcium influx, but also on the type of
agonist. Camphor, a waxy substance used topically for its
analgesic and counterirritant properties, activates TRPV1
independently of the vanilloid binding site and

Fig. 2. Tentative model of TRPV1 capsaicin-induced Ca2+-dependent desensitization. The cytosolic ankyrin-repeat domain of TRPV1
(residues 101-364) binds ATP and calmodulin in a Ca2+-dependent manner competes with ATP at the same interaction site (Lishko et al.
2007). These two competitors have opposite effects on TRPV1 channel activity: ATP sensitizes the channel whereas the Ca2+-CaM
complex inhibits it. A short C-terminal juxtamembrane segment adjacent to the TRP box (most likely around R701) is predicted to
contribute to the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and might be involved in the slowed gating
kinetics of the desensitized TRPV1 channel. The capsaicin molecule binds to the TRPV1 receptor at the channel-lipid interface between
S3 and S4. Ca2+-dependent activation of phospholipase C (PLC) results in the hydrolysis of localized PIP2 to 1,4,5-trisphosphate and
diacylglycerol (DAG), leading to inactivation of the TRPV1 channel. Acute desensitization could alter the Ca2+-dependent allosteric
coupling between the capsaicin binding site and the PIP2-sensitive gating apparatus of the TRPV1 channel. A high concentration of
intracellular ATP is required for PIP2 resynthesis by sequential phosphorylation of its precursor phosphatidylinositol by
phosphatidylinositol 4-kinases (PI4K) and phosphatidylinositol 4-phosphate 5-kinases (PIP5K).

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Acute Desensitization of TRPV1 S63


desensitizes it in a Ca2+-independent manner (Xu et al.
2005). Another natural product, piperine, the pungent
component of black pepper, has a higher degree of
cooperativity (Hill coefficient ~4) and a clear propensity
to cause greater desensitization of TRPV1 than capsaicin
(McNamara et al. 2005), even in the absence of Ca2+ in
the bath. It therefore
rearrangements in the TRPV1 channel complex include
not only the putative phosphorylation sites but also
several domains in or near the agonist-binding site or
sites that undergo conformational changes inactivating
the channel. In contrast to the rapid onset of the first
capsaicin response recorded in a 2 mM Ca2+-containing
bath solution, the time constant characterizing the
activation of the subsequent response is slower by about
one order of magnitude (Fig. 1A). This might be the
result of a decreased apparent affinity of desensitized
TRPV1 receptors for capsaicin, which is also supported
appears that structural
by the observation that [3H]RTX does not bind to
desensitized TRPV1 (Jung et al. 2004). Indeed, in the
presence of 2 mM Ca2+, desensitization of the
recombinant TRPV1 receptor after exposure to 1 μM
capsaicin can be overcome when its concentration is
increased to 30 μM (Fig. 3A).
A mutant TRPV1 channel S502A/T704I in
which two putative consensus sites for CaMKII are
simultaneously (but not individually) replaced with
nonphosphorylatable residues is insensitive to capsaicin
(Jung et al. 2004). However, another nonphospho-
rylatable mutant S502A/T704A is fully functional,
indicating the requirement for a specific residue at
position 704 (Novakova-Tousova et al. 2007). Moreover,
a point mutation at arginine residue R701 that, together
with T704, lies within a short C-terminal juxtamembrane
segment adjacent to the TRP box, strongly affected the
kinetics of capsaicin-evoked currents. As this residue

Fig. 3. (A) Capsaicin applied at high concentration (30 µM) reactivates TRPV1 channels that had previously been desensitized to 1 µM
capsaicin in Ca2+-containing bath solution (2 mM). (B) Changes in intracellular calcium concentration induced by consequent
applications of a heat ramp (20-48 °C in 30 s), 1 µM capsaicin and a temperature ramp (20-42 °C) applied in the presence of capsaicin,
monitored using ratiometric Ca2+ -imaging in the presence of 2 mM Ca2+ (n = 6 cells). For microfluorometric Ca2+ measurements,
TRPV1-expressing HEK293T cells were incubated in extracellular solution containing 2 µM FURA-2 AM for 45 min at 37 °C. (C, D, E)
The effects of acute Ca2+-dependent capsaicin-induced desensitization on the gating of TRPV1 by other modalities. (C) Time course of
TRPV1-mediated whole-cell currents induced by depolarizing voltage steps recorded at 25 °C in extracellular control solution (holding
potential -70 mV, voltage steps from -140 to +140 mV, increment +20 mV) before (open circles) and after (filled circles) desensitization
evoked by 1 µM capsaicin. Current-voltage relationship for currents obtained before and after acute desensitization such as shown in
the inset. (D) Whole-cell current responses to consecutive applications of a heat ramp (from 25 °C to 48 °C in 3 s) (a) and capsaicin
(1 µM and 30 µM) (b-e) in a Ca2+-containing bath solution. Consecutive currents recorded in response to heat ramps applied in a
standard bath solution (f), in the presence of 1 µM (g) and 30 µM (h) capsaicin. Current-temperature relationships for currents shown in
(a, f-h) demonstrate a strong potentiating effect of capsaicin on the heat-induced currents.