1,6 · 12.08.2013 · that the perioperative warming of surgical patients reduces postoperative...
TRANSCRIPT
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Complex modulation of TRPM8 cold receptor by volatile anaesthetics and role in
complications of general anaesthesia
Fabien Vanden Abeele1,5,7, Artem Kondratskyi1,5, Charlotte Dubois1,5, George
Shapovalov1, Dimitra Gkika1, Jérôme Busserolles4, Yaroslav Shuba2,3,6, Roman
Skryma1,6, and Natalia Prevarskaya1,6
1Inserm U1003, Equipe labellisée par la Ligue Nationale Contre le Cancer. Laboratory of
Excellence, Ion Channels Science and Therapeutics; Université des Sciences et Technologies
de Lille (USTL), Villeneuve d’Ascq, France.
2Bogomoletz Institute of Physiology and International Centre of Molecular Physiology of the
National Academy of Sciences of Ukraine, Kiev, Ukraine 3State Key Laboratory, Kiev, Ukraine 4Inserm UMR 1107, Clermont Université, Université d’Auvergne, Pharmacologie
Fondamentale et Clinique de la Douleur, Faculté de Médecine, Clermont-Ferrand, France
Running title: TRPM8 and complications of anaesthesia
Keywords: volatile general anaesthetics; TRPM8 cold receptor; dorsal root ganglion (DRG)
neurons; hypothermia; TRPM8-knockout mice
5These authors equally contributed to this work 6Share senior authorship 7To whom correspondence should be addressed: Fabien Vanden Abeele, PhD, Laboratoire de
Physiologie Cellulaire, Inserm U1003, USTL, Villeneuve d’Ascq, 59656 France; E-mail:
© 2013. Published by The Company of Biologists Ltd.Jo
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JCS Advance Online Article. Posted on 13 August 2013
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Summary
The mechanisms by which volatile general anaesthetics (VAs) produce a depression of
central nervous system are beginning to be better understood, but little is known about a
number of side effects. Here, we show that the cold receptor, TRPM8, is complexly
modulated by clinical concentration of VAs in dorsal root ganglion neurons and HEK-
293 cells heterologously expressing TRPM8. VAs produced transient enhancement of
TRPM8 via a depolarizing shift of its activation towards physiological membrane
potentials, followed by a sustained TRPM8 inhibition. Stimulatory action of VAs
engaged molecular determinants distinct from those used by the TRPM8 agonist.
Transient TRPM8 activation by VAs could explain such side effects as inhibition of
respiratory drive, shivering and cooling sensation during the beginning of anaesthesia,
whereas the second phase of VA action associated with sustained TRPM8 inhibition may
be responsible for hypothermia. Consistent with this, both hypothermia and inhibition
of respiratory drive induced by VAs are partially abolished in TRPM8-null animals.
Thus, we propose TRPM8 as a new clinical target for diminishing common and serious
complications of general anaesthesia.
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Introduction
Cellular membrane ion channels participate in all physiological processes ranging from
electrogenesis and the activation of numerous functional response(s) to gene expression and
to the determination of cell fate. They are also important determinants of numerous
pathological states and are the targets of therapeutic, as well as adverse actions of clinically
used pharmacological agents. Volatile general anaesthetics (VAs) induce safe and reversible
loss of consciousness, enabling surgical procedures to be carried out (Stachnik, 2006; Bovill,
2008; Franks, 2008). Several membrane ion channels and receptors have been identified as
targets of various VA action (Rudolph and Antkowiak, 2004; Hemmings et al., 2005).
Enhancement of the activity of inhibitory ionotropic receptors (Lobo and Harris, 2005; Zeller
et al., 2008), depression of the excitatory ionotropic receptors (Yamakura et al., 2001),
potentiation of the 2P-domain K+ channels (Patel et al., 2001), inhibition of voltage-gated Na+
channels (Hemmings, 2009) and of the transient receptor potential (TRP) family member,
TRPC5 (Bahnasi et al., 2008), probably all contribute to the central clinically relevant
anaesthetic effects, whereas the action of the same VAs on other ion channels may at least in
part be responsible for their numerous adverse effects (Stachnik, 2006; Bovill, 2008). Indeed,
activation by the “pungent” VAs (those that are known to excite peripheral nociceptive
neurons), isoflurane and desflurane of TRP-member, TRPA1 (Matta et al., 2008; Cornett et
al., 2008; Eilers et al., 2010) and sensitization of TRPV1 to its agonists, capsaicin and protons
(Cornett et al., 2008; Eilers et al., 2010), may contribute to postoperative pain and
inflammation, as well as producing airway irritation. Hypersensitivity to cold temperatures
and a decreased shivering threshold are also common complications observed after
administering VAs (Kurz et al., 1997; Kurz, 2008; Sessler, 2008), suggesting that at least
some of their representatives may sensitize peripheral cold receptors. Other peripheral effects
of these agents are still mechanistically poorly studied. Among these effects excitation by
VAs of mammalian nociceptor afferents (MacIver and Tanelian, 1990), hypersensitivity of
laryngeal C-fibers (Mutoh T and Tsubone, 2003), excessive respiratory reactions such as
coughing, inhibition of breathing (apnea), laryngospasm, and secretion (Drummond, 1993). In
fact, respiratory complications are the most frequent complications and the leading cause of
death during anaesthesia. Respiratory depression, laryngospasm, apnea and other causes
induce hypoxia. These complications are resolved spontaneously by correct treatment, but are
potentially lethal if they are not detected in time, or in case of inability to intubate or ventilate
the patient. Interestingly, several studies have reported that a cold air stimulus consistently
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cause an inhibition of breathing (Sekizawa et al., 1996) and that this inhibition of respiratory
drive can also be caused by menthol (Eccles, 1994), suggesting that at least some VAs may
sensitize peripheral cold receptors. anaesthetics are also implicated in perioperative
hypothermia (Sessler, 2008) by at least sensitizing peripheral thermoreceptors. Hypothermia
is a multifactorial, common and serious complication of anaesthesia and surgery manifested
by the drop of core body temperature by as much as 6º C. The combination of anaesthetic-
induced impairment of thermoregulatory control and exposure to a cool operating room
environment causes most surgical patients to become hypothermic. Mild intraoperative
hypothermia triples the occurrence of postoperative wound infections and myocardial events,
as well as increasing perioperative blood loss (Kurz, 2008). Furthermore, it delays
postoperative recovery and prolongs the effect of almost all anaesthetic drugs (Kurz, 2008).
This side effect could be reduced by perioperative warming, and recent evidence suggests that
perioperative warming may be given routinely to all patients of various surgical disciplines, in
order to counteract the consequences of hypothermia (Sajid et al., 2009). Overall, they show
that the perioperative warming of surgical patients reduces postoperative wound pain, wound
infection and shivering but does not entirely solve this problem (Sajid et al., 2009). As a
consequence, a better understanding of this multifactorial side effect could be very relevant in
medical practice.
Members of TRP-channel superfamily are the principal source of thermoreceptors of
various modalities (Schepers and Ringkamp, 2009). Of these, transient receptor potential
melastatin 8 (TRPM8), which is expressed in the subset of dorsal root ganglion (DRG) and
trigeminal sensory neurons, is activated by innocuous cold (<25° C) and chemical imitators of
cooling sensation such as menthol, icilin and eucalyptol (McKemy et al., 2002). The
activation of TRPM8 channel by cold or chemical agents depolarizes sensory nerve fibres
causing their excitation and transmission of the signal to the integrative centres of the CNS.
However, despite being proven to be a principal detector of environmental cold (Bautista et
al., 2007; Dhaka et al., 2007), the role of TRPM8 in the way in which VAs induce
complications to general anaesthesia, has yet to be assessed.
Results
Modulation of recombinant TRPM8 by VAs. Application of halothane (0.5 mM) to the
HEK-293 cells stably transfected with human TRPM8 (HEKM8 cells) activated a membrane
current with biophysical properties, such as a prominent outward rectification and close to 0
mV reversal potential, which is similar to the current activated by the established TRPM8
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stimulus, cold temperatures (Fig. 1A). TRPM8 activation by halothane did not increase
monotonically with concentration, but exhibited bell-shaped concentration dependence (Fig.
1B): at room temperature (23°C) anaesthetic potently activated TRPM8 in the narrow
concentration range of 0.20-1 mM, with 0.5 mM being the most effective. Comparison of the
steady-state activation curves of the background membrane current carried through TRPM8
channels (ITRPM8) in HEKM8 cells at room temperature and ITRPM8 over-activated by further
exposure to halothane showed, that anaesthetic produces a depolarizing shift in the voltage-
dependence of the TRPM8 channel activation by about 100 mV (Figs1C, D), which may
underlie its agonistic action mechanism.
As far as other VAs used in clinical practice are concerned, clinically-relevant
concentrations of isoflurane (0.2 mM, n=5) (Fig. 2A), desflurane (0.2 mM, n=3) (Fig. 2B),
and sevoflurane (0.2 mM, n=3) (Fig. 2C) also rapidly activated a membrane current in HEKM8
cells with similar biophysical properties to the one activated by halothane. Since either cold
stimulus, menthol, or any of the VAs tested were unable to induce such a current in the
control HEK-293 cells (HEKM8 cells without tetracycline induction, HEKCtrl cells), we
concluded that in HEKM8 cells, this is associated with TRPM8 activation (ITRPM8).
TRPM8 is a non-selective, Ca2+-permeable cationic channel, therefore its activity in
response to VAs can be evaluated by measuring the cytosolic calcium concentration ([Ca2+]c).
Calcium imaging experiments using calcium probe fura-2AM, clearly demonstrated that
isoflurane (0.5 mM, n=9) induces a pronounced elevation of [Ca2+]c i.e. up to 1 µM in HEKM8
cells, but is ineffective in control HEKCtrl cells. This would suggest that this elevation most
likely results from isoflurane-stimulated, TRPM8-mediated Ca2+ entry (Fig. 2E).
Interestingly, withdrawal of isoflurane led not only to the recovery of [Ca2+]c in HEKM8 cells,
but also to the substantial decrease in their basal room temperature [Ca2+]c level from the pre-
isoflurane value of 430±75 nM, to the post-isoflurane value of 205±7 nM (n=9) (Fig. 2E). We
also performed additional calcium imaging experiments (at 37°C) with fura-2 in which the
effect of TRPM8 inhibitor (BCTC 10 µM) on isoflurane (0.5 mM) induced sustained calcium
entry was assessed. This calcium entry was effectively antagonized by the TRPM8 inhibitor
BCTC (Fig. 2F).
To more thoroughly investigate how TRPM8 activity is affected by general anaesthetics,
we performed measurements of single-channel activity of the channel. High expression level
of TRPM8 protein in HEKM8 cells led to the presence of multiple channels in all acquired
patches and limited our analysis to characterization of overall probability of TRPM8 opening
(NPopen). Fig. 2G and H compare basal and isoflurane stimulated (0.2 mM, n=5) TRPM8
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activity by analyzing fragments before and 20 sec after isoflurane application and shows a
significant increase in TRPM8 NPopen upon stimulation by isoflurane.
Dual action of VAs on recombinant TRPM8. Stimulatory action of VAs on TRPM8 was
followed by its sustained inhibition. As shown in Fig. 3, initial activation of ITRPM8 in HEKM8
cells in response to clinically relevant concentrations of halothane (Fig. 3A), isoflurane (Fig.
3B), desflurane (Fig. 3C) and sevoflurane (Fig. 3D) was followed by the profound current
decrease despite a continuing presence of the drug. This decrease constituted 75% to 85% of
the maximally stimulated value (Fig. 3E) and commonly brought the current down to the
steady-state level, or even below the baseline ITRPM8 at room temperature. Chloroform
(CHCl3) also produced a delayed sustained ITRPM8 inhibition, but its extent was notably
smaller than the other VAs studied (i.e., 40%, Fig. 3E). Moreover, incubation of HEKM8 cells
in 0.25 mM CHCl3 for 15 min, resulted in about a 40% decrease in the amplitude of basal
ITRPM8 at room temperature (Fig. 3F). This is consistent with the notion that prolonged action
of VAs induces TRPM8 inhibition.
Prolonged exposure to VAs was able to inhibit, not only background non-stimulated
TRPM8 activity at room temperature, but also stimulated activity. Fig. 3G shows that
exposure of HEKM8 cells to isoflurane at 37°C, besides activating a TRPM8-mediated [Ca2+]c
rise, at the same time diminished the response to subsequent cold stimulus, whereas without
isoflurane, a series of cold stimuli produced ever-increasing [Ca2+]c responses (Fig. 3H).
Taken together, these data clearly demonstrate a dual action of VAs on TRPM8: a rapid and
transient activation followed by delayed sustained inhibition.
The mechanism of the VA action on recombinant TRPM8.
Our results clearly show that the rapid TRPM8-activating mode of the VAs' action is
associated with a depolarizing shift in the voltage-dependence of the channel activation (Figs1
C, D). Thus, we next asked what kind of molecular interactions might underlie this
phenomenon. First, there might be a direct interaction of the VAs with channel protein, as was
suggested for the mammalian K2P channels, TREK-1 and TASK (Patel et al., 1999). In the
case of TREK-1 and TASK channels, their C-terminal regions were found to be critical for
anaesthetic-induced activation (Patel et al., 1999). Second, a TRPM8 channel may change its
gating due to mechanical tension coming from the altered membrane micro-curvature as a
result of the partitioning of highly hydrophobic VAs into the lipid bilayer. Such a mechanism
has been suggested for the action of a number of lipid modifiers on the mechano-gated TREK
and TRAAK K2P channels (Patel and Honore, 2001). Third, VAs may influence the sensitivity
of a TRPM8 channel to its endogenous modulators, first of all to phosphatidylinositol
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biphosphate (PIP2), which regulates the activation of TRPM8 through interaction with the
channel’s TRP domain (Rohács et al., 2005).
To test the possibility that the site of VA’s interaction with TRPM8 channel may coincide
with the one for its classical agonist menthol, we used two channel mutants, Y745H and
L1009R, which have been shown to have strongly reduced menthol and icilin, but not cold,
voltage or PIP2 sensitivity (Bandell et al., 2006). As shown in Fig. 4A, both halothane and
menthol are capable of activating ITRPM8 in HEK-293 cells transfected with the wild-type
TRPM8. However, in HEK-293 cells transfected with Y745H (n=3) and L1009R (n=3)
mutants, exposure to menthol failed to activate any measurable current, whilst current
activation by halothane was not affected (Figs 4B, 4C). Thus, the molecular determinants,
within a TRPM8 channel, required for channel gating by VAs, are distinct from those used by
menthol.
To verify the “bilayer-coupled” hypothesis, we used a lipid bilayer modifier, namely
arachidonic acid (AA), which is a polyunsaturated fatty acid with proven efficacy on
mechano-sensitive K2P channels. By partitioning into the outer leaflet of the lipid bilayer, AA
is known to induce a convex membrane curvature (i.e. membrane crenation), leading to the
activation of TREK and TRAAK channels (Patel and Honore, 2001). In our hands, TRPM8
opened either by menthol or by VAs, was not mimicked by AA (10 µM; n=5) (Fig. 5A). We
also failed to detect any TRPM8 activation by anionic amphipath trinitrophenol (TNP), which
has proven efficacy on mechano-sensitive K2P and TRPA1 channels (Patel and Honore, 2001,
Hill and Schaefer, 2007). Moreover, consistent with previous studies (Andersson et al., 2007,
Bavencoffe et al., 2011), AA produced ITRPM8 inhibition, rendering the bilayer-coupled
activation hypothesis for the anaesthetic action on TRPM8 quite unlikely.
In our previous study, we showed that cationic amphipaths, such as chlorpromazine
(CPZ), which preferentially inserts into the inner leaflet of the bilayer, thereby facilitating the
formation of the membrane cup-shapes (cup-formers), is able to antagonize the activating
effects of menthol and lysophospholipids on TRPM8 (Vanden Abeele et al., 2006). When
CPZ (1 µM) was applied together with CHCl3, it not only prevented CHCl3-mediated TRPM8
activation, but also reversibly inhibited baseline ITRPM8 at room temperature (Fig. 5B). Similar
antagonistic actions of cup-formers have been reported for the mechano-gated K2P channels
(Patel and Honore, 2001). Thus, we cannot entirely rule out possible TRPM8 modulation by a
bilayer alteration mechanism, particularly with respect to the inhibitory influence of
membrane invaginations on TRPM8 channel activation. However, it is unlikely that this
mechanism underlies VA action. Given that modulation of TRPM8 by VAs correlates with
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their ability to partition into the membrane (halothane/sevoflurane > desflurane) one cannot
also exclude VAs signalling via membrane fluidity.
Next, we examined a possible dependence of the VA-activated TRPM8-mediated current
on the PIP2 regulatory system, which is important for supporting TRPM8 channel function
(Rohács et al., 2005). After establishment of whole-cell configuration, TRPM8-mediated
current usually runs down, due to the depletion of PIP2 by lipid phosphatase. This rundown
could be accelerated by intracellular polylysine dialysis, which acts as a PIP2 scavenger
(Rohács et al., 2005). In our experiments, inclusion of 3 µg/ml polylysine in the pipette
solution within 15-25 minutes of cell dialysis, inhibited more than 80% of the cold-activated
ITRPM8. However, application of CHCl3 (Fig. 5C) or halothane (Fig. 5D) was still able to
reactivate the current (n=3), thus suggesting that agonistic action of VAs on TRPM8 occurs
through a mechanism distinct from that described for menthol, temperature or icilin, since it
seems to be independent of the PIP2 levels.
Modulation by VAs of endogenous TRPM8 in DRG neurons.
Next we studied the effects of halothane on endogenous TRPM8 in isolectin B4(IB4)-
negative small diameter DRG neurons (see Methods section). These are known to
preferentially express the TRPM8 cold receptor, (but not theTRPV1 heat receptor and only in
negligible levels of the TRPA1 receptor of pungent compounds (Kobayashi et al., 2005).
Since VAs can potentially modulate or directly activate different types of endogenous
channels, we first evaluated the ability of VAs to specifically affect menthol-activated current
in DRG neurons. Fig. 6A shows that the application of halothane (0.5 mM) on top of menthol
(100 µM) potentiates menthol-activated ITRPM8 by about 30% at 100 mV and by about 50% at
-100 mV (n=5). The current in the presence of halothane had the same reversal potential and
apparent rectification as the menthol-activated one (Fig. 6B), suggesting that current
stimulation occurs due to the anaesthetic’s action on TRPM8 channels and not on some other
channel type(s). When neurons were clamped at a holding potential (Vh) close to the neurons’
resting potential (i.e. -60 mV), application of menthol (100 µM) activated an inward current
(Fig. 6C; n=3), which was sufficient to produce depolarization and action potentials (AP)
firing which were readily detectable in the current-clamp mode (Fig. 6D; n=4). Application of
halothane on top of menthol augmented the inward current (Fig. 6C; n=3), further depolarized
the neuron and substantially increased the frequency of AP firing (Fig. 6D; n=4), which is
consistent with the ability of halothane to enhance TRPM8-mediated signalling.
In a second series of experiments, we examined whether VAs not only potentiate menthol-
activated current, but also directly activate TRPM8 in IB4-negative small diameter DRG
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neurons. Fig. 7A shows representative recordings of the background currents at different step
potentials in DRG neuron and the currents following exposure to halothane (0.5 mM). One
can see that application of halothane alone was sufficient to activate membrane current above
the baseline (Fig. 7A, B) and this current had biophysical properties similar to that activated
by menthol (compare I-Vs of Fig. 6B and Fig. 7C). In our hands isoflurane (0.5 mM) also
activated membrane current with biophysical properties similar to ITRPM8. Moreover, this
current was effectively antagonized by the TRPM8 inhibitor BCTC (10µM) (suppl. Fig. 7).
Current activated by halothane in IB4-negative, menthol-responding DRGs, could be
suppressed by the known TRPM8 inhibitor, capsazepine (20 µM, Fig. 7D). Given that of two
other VA-sensitive TRP members, TRPV1 and TRPA1 (Matta et al., 2008; Cornett et al.,
2008; Eilers et al., 2010), the former is not expressed in these neurons, whereas the latter is
not sensitive to capsazepine, this provides additional assurance of the involvement of TRPM8
in the observed effects (Simon and Liedtke, 2008). Moreover, as in HEKM8 cells, prolonged
exposure of menthol-responding DRG neurons to halothane after initial current activation,
caused pronounced current inhibition (Fig. 7E), indicating that VAs exert dual action not only
on recombinant TRPM8, but also on the native neuronal one as well. Calcium imaging
experiments with fura-2AM (at 37°C) demonstrated that halothane (0.5 mM, n=5) also
induces transient [Ca2+]c increase in menthol-sensitive DRG neurons (Fig. 7F). The fact that
in the current-clamp mode, a brief application of halothane (0.5 mM, n=3) alone was able to
cause depolarization and AP firing (Fig. 7G), that was inhibited by the TRPM8 antagonist
BCTC (10μM), demonstrates the physiological significance of VA action on the TRPM8 cold
receptor in sensory neurons. Further, to exclude the possibility of possible involvement of
TRPA1, we assessed the effect of halothane (0.5 mM) on membrane currents in DRG neurons
in the presence of TRPA1 inhibitor HC-030031. In these conditions halothane also activated
membrane current with biophysical properties similar to ITRPM8 suggesting independence of
TRPA1 (Fig. 7I). Additionally, halothane activated current was effectively inhibited by BCTC
(10μM), again confirming the involvement of TRPM8 (Fig 7H).
Reduction of common complications of general anaesthesia: hypothermia and
inhibition of respiratory drive induced by VAs in TRPM8-null animals.
The involvement of TRPM8 in the mechanism(s) of VA-induced complications of general
anaesthesia has yet to be evaluated. On the one hand, it has been well established by several
studies that a cold air or menthol stimulus consistently caused an inhibition of respiratory
drive (Eccles, 1994; Sekizawa et al., 1996) and, on the other hand, that a pharmacological
blockade of TRPM8 produces hypothermia (Knowlton et al., 2011; Almeida et al., 2012). We
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hypothesized that transient TRPM8 activation by VAs may be at least partly responsible for
the inhibition of respiratory drive, whereas delayed sustained inhibition of TRPM8 following
VA administration, may contribute to the induction of hypothermia. To test this hypothesis,
we conducted comparative functional tests on wild-type and TRPM8-knockout mice.
Figs8A and 8B show a comparison of the body temperatures and respiratory frequency of
the wild-type and TRPM8-/- mice during isoflurane anaesthesia. Body temperature and
respiratory frequency of the wild-type and TRPM8-/- mice were not statistically different at
resting state: 38.9°C±0.2 in wild-type animals versus 39.3°C±0.2 in TRPM8-/- mice (Student’s
t-test; p>0.05 for both). Following exposure to isoflurane both functional parameters
underwent an essential reduction in both animal groups, although the reduction was more
pronounced in the wild-type animals. The body temperature of TRPM8-/- mice remained
statistically significantly higher (Student’s t-test; ***p<0.01; n=10) during the whole 20 min
monitoring period, consistent with the notion that the TRPM8 cold receptor contributes to the
lowering of body temperature in response to isoflurane. On the other hand, the respiratory
frequency in TRPM8-/- mice was statistically significantly higher (Student’s t-test; ***p<0.01;
n=10) only during the first half of the 20-min-long monitoring period. During the second half,
no statistically significant differences were detected, suggesting a role for the TRPM8 cold
receptor in this complication only at the beginning of anaesthesia. These results agree
perfectly with previous reports showing that a cold air or menthol stimulus cause an inhibition
of respiratory drive (Eccles, 1994; Sekizawa et al., 1996) and with our current findings that
VAs produce a transient activation of the TRPM8 cold receptor. Indeed, we demonstrate that
VAs as well as activating TRPM8 also induce its strong desensitization to the physiological
agonist, cold (Figs 3G, H). Such desensitization may be viewed as an equivalent to
pharmacological TRPM8 blockade, which was recently reported to evoke a decrease in body
temperature in rats and mice (Knowlton et al., 2011; Almeida et al., 2012) and, thus, can
explain hypothermia observed in our experiments.
Discussion
In the present study, we show for the first time that VAs, including the archetypal
anaesthetic chloroform as well as halothane, isoflurane and modern anaesthetics such as
desflurane and sevoflurane activate both the heterologously expressed and the endogenous
TRPM8 cold receptor. Activation of endogenous TRPM8 in rat DRG neurons by halothane
results in depolarization and increased firing, thereby indicating induction of peripheral cold
sensitivity by VAs. Our results suggest that: 1) TRPM8 modulation by a bilayer alteration
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mechanism cannot account for the agonistic actions of the VAs; 2) The molecular
determinants required for channel gating by VAs are distinct from those used by menthol; 3)
The agonistic action of VAs on TRPM8 occurs through a distinct mechanism from the one
described for menthol, temperature or icilin, since it seems to be independent of the PIP2
levels.
Recent ultra-structural studies on general anaesthetic-sensitive bacterial homologues of
mammalian Cys-loop-type ionotropic receptors, have, at least for this type of receptors,
revealed a common general- anaesthetic binding site. This site consists of mainly hydrophobic
amino acids located within the cavity on the extracellular side of the receptor-forming
subunits (Nury et al., 2011). The extent to which structural considerations derived from Cys-
loop-type ionotropic receptors can be applied to TRP channels remains to be seen.
In addition to inducing unconsciousness, amnesia and analgesia VAs' also cause a number
of side-effects, which include an excitation of mammalian nociceptor afferents (MacIver and
Tanelian, 1990), hypersensitivity of laryngeal C-fibres (Mutoh and Tsubone, 2003), excessive
respiratory reactions such as coughing, inhibition of breathing (apnea), laryngospasm and
secretion (Drummond, 1993). Hypersensitivity to cold temperatures and a lower shivering
threshold are also common complications observed after administering VAs (Kurz et al.,
1997; Kurz, 2008; Sessler, 2008; MacIver and Tanelian, 1990), suggesting that at least some
of their representatives may sensitize peripheral cold receptors. Our results establish a link
between cold sensation and VA administration, since we show that VAs directly activate the
TRPM8 cold receptor.
Recently, it has been shown that “pungent” inhalation general anaesthetics, (which are
known to excite peripheral nociceptors), namely isoflurane and desflurane, activate TRP-
member TRPA1 and sensitize the TRPV1 heat receptor to its agonists, capsaicin and protons
(Cornett et al., 2008; Matta et al., 2008; Eilers et al., 2010), but do not affect the TRPM8 cold
receptor. These authors concluded that the pro-nociceptive effects of these anaesthetics
combined with surgical tissue damage may specifically contribute to postoperative pain and
inflammation, as well as producing airway irritation. In our hands, TRPM8 was observed to
be activated with quite similar potency by non-pungent VAs, halothane, chloroform and
sevoflurane, as well as the pungent ones, isoflurane and desflurane. TRPM8 activation by
these compounds was validated in HEKM8 cells as well as DRG neurons using both patch-
clamp and calcium imaging techniques, which together strongly confirm our observations. We
currently have no explanation why our results on TRPM8 sensitivity to the VAs are different
from those reported earlier (Matta et al., 2008). However, we maintain that aside from being
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able to excite TRPA1/TRPV1-expressing nociceptors, VAs are also capable of exciting a
separate population of TRPM8-expressing cold-sensitive afferents, and this may contribute to
the set of complications associated with impaired peripheral sensitivity to cold temperatures.
Concerning perioperative hypothermia, it has been demonstrated that TRPM8 agonists
such as, menthol, icilin and 1.8-cineole enhance thermogenesis (Masamoto et al., 2009),
whilst TRPM8 antagonists induce the decrease in body temperature (Knowlton et al., 2011;
Almeida et al., 2012). However, in view of possible non-specific side effects of these
compounds, attribution of their thermogenicity solely to TRPM8 should be taken with
caution. Our functional tests on wild-type and TRPM8-knockout mice clearly show that a lack
of TRPM8 reduces hypothermia associated with isoflurane anaesthesia and that these effects
are probably linked to the strong desensitization of TRPM8 by VAs to the physiological
agonist, cold. Such desensitization may be viewed as equivalent to pharmacological TRPM8
blockade, and thus, can explain the hypothermia observed in our experiments. Although more
studies are necessary to explore the role of other thermo-receptors (i.e. TRPA1, TRPV1,
TREK) in VA-induced hypothermia, these data unequivocally demonstrate that the VA-
mediated activation of TRPM8 is, at least partially, involved in this process.
The relation between respiratory depression and TRPM8 modulation by VAs appears
more complex. Similarly, respiratory depression by anaesthetics is also multifactorial.
Respiratory complications are the most frequent complications and the leading cause of death
during anaesthesia. This depressant effect on respiration by VAs and isoflurane, in particular,
is enhanced by premedication and concomitant administration of other preparations which
also have a depressing effect on respiration. In medical practice, it is therefore essential to
closely monitor breathing and to support it, where necessary, by mechanical ventilation.
Patients with myasthenia gravis are particularly susceptible to preparations which cause
respiratory depression. These complications are resolved spontaneously by correct treatment,
but can be potentially lethal if they are not detected in time or if there is an inability to
intubate or ventilate the patient. Interestingly, several studies have reported that, on the one
hand, a cold air stimulus consistently caused an inhibition of breathing (Sekizawa et al., 1996)
and, on the other hand, that inhibition of respiratory drive can also be caused by menthol
(Eccles, 1994). It has also been demonstrated that a subpopulation of airway vagal afferent
nerves express TRPM8 receptors and that activation of TRPM8 receptors by cold excites
these airway autonomic nerves increasing airway resistance (Xing et al., 2008). These data
suggest the involvement of TRPM8 activation in the respiratory drive inhibition. Our
findings, that in wild-type mice, the progression of isoflurane anaesthesia is accompanied by a
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more pronounced decrease in respiratory frequency than in the TRPM8-knockout ones, are
fully consistent with this notion. Our data clearly show that the difference between wild-type
animals and TRPM8-/- mice was statistically significant during the first half of the monitoring
period. During the second half, no statistical differences were found, suggesting a role for
TRPM8 cold receptor activation at the beginning of anaesthesia. These observations correlate
with the dual effect of VAs on TRPM8. At the same time, our results demonstrate that
TRPM8 is not the only target implicated in this process, since the depressant effect of
isoflurane on respiratory frequency is partially preserved in KO mice. We have to bear in
mind that respiratory depression by anaesthetics is also multifactorial, and that the TRPA1
channel could be also involved, given the evidence that desflurane induces airway contraction
via an activation of TRPA1 in sensory C-fibres (Satoh and Yamakage, 2009).
In conclusion, our results provide a better understanding of the side-effects of volatile
anaesthetics, thereby contributing to the public health domain in general and in particular to
the elaboration of new anaesthetic strategies.
Materials and Methods
Cells– Studies of recombinant TRPM8 were performed on HEK-293 cells stably transfected
with human TRPM8 under the control of a tetracycline-inducible promoter (HEKM8 cells)
(Thebault et al., 2005). HEKM8 cells without tetracycline induction were used as controls
(HEKCtrl cells). Neurons were isolated from the lumbar DRG of adult Wistar rats (250-300 g)
using an enzymatic digestion procedure. Cell suspension was plated onto Petri dishes filled
with 10 % fetal calf serum- and 8 µg/ml gentamicine-supplemented DMEM (“Gibco”, UK)
culture medium and incubated for 18-24 h at 37° С in the 95 % air 5 % СО2 atmosphere,
prior to use in electrophysiological experiments. Immediately before electrophysiological
experiments, neurons were stained with 10 µg/ml IB4-Alexa Fluor 488 (Molecular Probes) for
20 min and then rinsed for 10 min in extracellular solution. Only IB4-negative small-diameter
neurons were used for subsequent experiments.
Electrophysiology and Solutions– Membrane currents were recorded in the whole cell
configuration of the patch clamp techniques using the Axopatch 200B amplifier (Molecular
Devices, Union City, CA). The resistance of the patch pipettes fabricated from borosilicate
glass capillaries (World Precision Instruments, Inc., Sarasota, FL) when filled with the
intracellular solution, was 2-3 megaohms for the whole cell recordings. In the whole cell
experiments, series resistance was compensated for by about 70%. Currents were filtered at 1
or 2 kHz and sampled at 10 kHz.
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Whole cell currents were measured under nearly symmetrical ionic conditions (i.e.
standard extracellular solution containing (in mM): 150 NaCl, 1 MgCl2, 5 glucose, 10
HEPES, pH 7.3 (adjusted with NaOH), whereas the pipette was filled with the intracellular
solution containing (in mM): 150 NaCl, 3 MgCl2, 5 EGTA, 10 HEPES, pH 7.3 (adjusted with
NaOH). For temperature control and solution exchange, the TC1-SL25 system (Bioscience
Tools, San Diego, CA) was used with the temperature probe placed near the patch pipette tip.
The system provided a temperature stability of at least 0.2°C. All experiments were carried
out at 23°C. During whole-cell patch-clamp recordings, DRG neurons were bathed in the
standard extracellular solution (in mM): 140 NaCl, 5 KCl, 5 glucose, 10 HEPES,
2 CaCl2, 1 MgCl2, pH 7.4, while dialyzed with Cs+-based intracellular patch-pipette solution
containing (in mM): 140 CsCl, 5 EGTA, 10 HEPES, 2 Mg-ATP, 0.5 Li-GTP, pH 7.3. For
current clamp recordings Cs+ was substituted with K+ in pipette solution.
Single-channel recordings were performed in cell-attached patches to HEKM8 cells stably
expressing TRPM8 protein. Cells were immersed in high-KCl solution, in order to bring cell
potential close to zero (in mM): 150 KCl, 2 MgCl2, 1 CaCl2, 5 glucose, 10 Hepes, PH 7.3
(adjusted with KOH) and the pipettes were filled with standard extracellular solution
described above. Recording of basal activity of TRPM8 was followed by application of 0.2
mM isofluorane and fragments before and 20 sec after application of isofluorane were
analyzed with the help of Clampfit-10 program (Molecular Devices, Union City, CA). The
obtained NPopen values were normalized to the basal NPopen levels individually for each
trace.
Calcium imaging experiments- These experiments were performed using the membrane-
permeable Ca2+-sensitive dye fura-2AM, as detailed previously (Vanden Abeele et al., 2006).
Reagents and preparation of anaesthetics- Halothane, chloroform and diethyl-ether were
purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France); isoflurane from
Laboratoires Belamont (Neuilly Sur Seine, France); sevoflurane from Abbott (Rungis,
France) and desflurane from Baxter (Maurepas, France). Other reagents were purchased from
Sigma-Aldrich. Saturated stock solutions of VAs were prepared in gas-tight bottles by
dissolving excess anaesthetic agents in bath solutions and stirring vigorously overnight. From
these stock solutions, fresh dilutions were made up immediately prior to experiments.
Data Analysis– Membrane current recordings obtained from cells were analyzed and plotted
using the pCLAMP 9 (Axon Instruments, Inc.) and Origin 5 software (Microcal Software
Inc.). Results were expressed as the means ± S.E. Statistical analysis was performed using
Student's t test (differences considered significant when p was <0.05).
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Acknowledgements
We are grateful to D. Julius for providing us with the Trpm8 knockout mice. We are grateful
to A. Patapoutian for providing us with the mutants Y745H and L1009R. This work was
supported by grants from INSERM, la Ligue Nationale contre le cancer, INTAS 05-1000008-
8223 and F46.2/001 (YS). Y.S. was in part supported by the visiting scientists program of the
Université des Sciences et Technologies de Lille. Artem Kondratskyi was supported by
fellowship from Fondation de la Recherche Médicale.
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Figure legends
Fig. 1. Halothane activates TRPM8 cold receptor heterologously expressed in HEK-293 cells.
(A) Original recordings of the baseline (acquired at point 1 of the time-course) and halothane-
activated (0.5 mM) (acquired at point 2 of the time-course) ITRPM8 in response to pulse
protocol with voltage-ramp portion shown above the recordings. (B) TRPM8 activation by
halothane was not dose dependent but exhibits a bell-shaped curve (Currents at +100 mV).
(C) Examples of the baseline and halothane-activated ITRPM8 in response to the depicted
voltage-clamp protocol, which were used to measure voltage-dependence of TRPM8 channel
open probability (Po, panel D); arrows in C point to the ITRPM8 tail currents at +60 mV, which
normalized amplitude as function of conditioning depolarizing pulse (ranging from -120 to
+160 mV) corresponds to the apparent Po (mean±s.e.m., n=4-6). Smooth curves in D –
Boltzmann functions fitted to the experimental data points; parameters of the fits, half
activation potential V1/2 and slope factor k, for the baseline (filled symbols) and halothane-
activated (open symbols) ITRPM8 are shown near respective curves.
Fig. 2. General volatile anaesthetics activate TRPM8 cold receptor. (A, B, C) Representative
recordings of the baseline current and currents after consecutive applications of clinically-
relevant concentration of isoflurane (0.20 mM; Fig. 2A; n=5), desflurane (0.20 mM; Fig. 2B;
n=3) and sevoflurane (0.2 mM, Fig. 2C; n=3) activate ITRPM8 above its baseline level at room
temperature (23°C) in HEKM8 cells. (D) Mean values of the ITRPM8 current recorded
immediately after anaesthetic application (Currents at +100 mV). (E) Application of
isoflurane (0.5 mM, n=9) induced sustained calcium entry up to 1µM in HEKM8 cells but was
ineffective in control HEK-293 cells at room temperature. Calcium imaging experiments were
made using the cytosolic calcium probe fura-2. (F) Calcium imaging experiments (at 37°C)
with fura-2. The effect of TRPM8 inhibitor (BCTC 10 µM) on isoflurane (0.5 mM) induced
sustained calcium entry was assessed. (G) Sample single channel activity of TRPM8 channel
in cell-attached patches. Basal activity is shown, followed by application of isoflurane (0.20
mM) as indicated by vertical arrow. The insets show higher-resolution fragments of TRPM8
activity before and after isoflurane application. (H) The barplot summarizes changes in Npopen
upon isoflurane application. ** indicates significant difference with p < 0.01, (n=5).
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Fig. 3. Dual action of general volatile anaesthetics on TRPM8 cold receptor during prolonged
application. (A, B, C, D) Representative time-courses of membrane current (measured at +100
mV, at 23°C) demonstrating that clinically-relevant concentration of halothane (Fig. 3A; n=5),
isoflurane (Fig. 3B; n=5), desflurane (Fig. 3C; n=3) and sevoflurane (Fig. 3D; n=3) induce
biphasic response in HEKM8 cells. Secondly; GAs induce strong inhibition of ITRPM8. (E)
Mean values of the residual membrane current (from similar experiments presented in A, B, C
and D) during GAs application. (F) Mean values of the ITRPM8 current recorded immediately
after establishment of the Whole Cell configuration (n=6). (G, H) Ca2+
imaging experiments
with repeated application of cold perfusion (33°C) (n=12) or in combination with isoflurane
(n=10) in HEKM8 (*p<0,05).
Fig. 4. Mechanism of activation of recombinant human TRPM8 in HEKM8 cells.
Representative time-courses of membrane current showing that halothane (0.5 mM) and
menthol (250 µM) activate ITRPM8 above the baseline at room temperature (23°C) in cells
transfected with the wild-type TRPM8 (TRPM8wt, panel A; n=6) and that menthol, but not
halothane loses the ability to activate current in the cells transfected with TRPM8 Y745H
mutant (panel B; n=3) that impairs sensitivity to menthol and icilin, but not to cold, voltage or
PIP2.
Fig.5. Mechanism of activation of recombinant human TRPM8 in HEKM8 cells. (A)
Representative time-course of membrane current showing that arachidonic acid (AA, 10 µM)
did not activate membrane current (n=5). (B) Cationic amphipath, chlorpromazine (CPZ, 1
µM) co-applied with chloroform (CHCl3, 2.5 mM) inhibits baseline ITRPM8 at room
temperature (23°C) and prevents its further activation by CHCl3, whereas CHCl3 alone is able
to activate current above the baseline (n=5). (C, D) Representative time-courses of membrane
current showing that intracellular dialysis of polylysine (Poly-L, 3 µg/ml) induces a rapid run-
down of ITRPM8 but does not interfere with the ability of CHCl3 (2.5 mM) or halothane (0.5
mM) to activate residual ITRPM8 above the baseline at room temperature (23°C) (n=3 for each).
Mean values (±s.e.m., n=3, in %) of the ITRPM8 current recorded immediately after CHCl3
(225%±14) or halothane application (171%±9). In all panels current was measured at
Vm=+100 mV.
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Fig. 6. Modulation by halothane of endogenous TRPM8 in isolectin B4 (IB4) negative small
diameter DRG neurons. (A) Representative recordings of the baseline current in DRG neurons
at 32° С and currents after consecutive applications of menthol (100 µM) and menthol plus
halothane (0.5 mM); voltage-clamp protocol is shown above the recordings; the inset
represents averaged normalized time-courses of the outward (at +100 mV) and inward at (at -
100 mV) current in 5 DRG neurons at +100 mV during application of menthol (100 µM) and
halothane (0.5 mM); current amplitudes were normalized to the immediate pre-halothane
values at respective potential before averaging (mean±s.e.m., n=5). (B) I-V relationships of
the currents in the presence of menthol (100 µM, open symbols) and menthol plus halothane
(0.5 mM, filled symbols) derived from ramp portions of the recordings presented in A. (C)
Potentiation by halothane (0.5 mM) of the inward menthol-evoked ITRPM8 at holding potential
Vh=-60 mV (n=3). (D) Enhancement by halothane (0.5 mM) of menthol-evoked action
potential (AP) firing in DRG neurons; the recording of neuron’s membrane potential was
performed in the current-clamp mode; application of menthol (100 µM) caused depolarization
and AP firing, whilst application of halothane on top of menthol caused further depolarization
and increase in the frequency of AP firing (n=4).
Fig. 7. Direct activation by halothane of endogenous TRPM8 in isolectin B4 (IB4) negative
small diameter DRG neurons. (A) Comparison of the steady-state activation curves of the
background (baseline) membrane current recorded in DRG neurons at room temperature and
current overactivated by further exposure to halothane alone (0.5 mM). (B) Mean values of
the background (baseline) membrane current at room temperature and current overactivated
by halothane (0.5 mM; n=5; *p<0.05). (C) I-V relationships of the baseline currents (open
symbols) or in the presence of halothane (0.5 mM; n=5; filled symbols). (D) Currents
recorded in DRG neurons and activated by halothane (1 mM) were inhibited by an inhibitor of
TRPM8, capsazepine (20 µM) (n=3). (E) Dual action of halothane on endogenous TRPM8.
Representative time-course of membrane current (measured at +100 mV, at 23°C). (F)
Calcium imaging experiments (at 37°C) with fura-2 demonstrated that halothane (0.5 mM;
n=5) induced significant calcium entry in menthol responding DRG neurons. (G) Activation
by halothane (0.5 mM) of action potential (AP) firing in DRG neurons is inhibited by BCTC
(10 µM) (n=3). The recording of neuron’s membrane potential was performed in the current-
clamp mode. (H, I) Halothane (0.5 mM) activates BCTC sensitive membrane currents in DRG
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neurons in the presence of TRPA1 inhibitor HC-030031. Representative time-course of
membrane current as well as current traces are shown.
Fig. 8. Hypothermia and inhibition of respiratory drive induced by VAs in TRPM8-null
animals. Statistically significant differences in body temperature (A) and respiratory
frequency (B) occurred between control and TRPM8-null mice (KO-TRPM8) during general
anaesthesia by isoflurane (1,25%). (Student’s t-test; ***p<0.01; n=10).
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C D
B A
1
2
3
4
n=5
n=5n=6
n=7
halothane, mM
10.
750.5
0.37
50.
25
I/Ic
ontr
ol
n=8
0 0
100
-100
1
200 ms
100
0 p
A
mV
baseline, 23o C
+halothane2
Figure 1.
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0 60 120 180 240 3000,0
0,5
1,0
1,5
2,0
2,5
3,0 HEK-293 M8+BCTC 10µM
HEK-293 M8
ratio
, 3
40
/38
0
Time, s
isoflurane 0.5 mM
0 60 120 180 240 300 3600
200
400
600
800
1000
1200
Time, s
HEK-293 M8
HEK-293 WT
[Ca
2+]c
, nM
isoflurane 0,5 mM
1
2
3
4
sevofluranedesfluraneisoflurane
n=3
n=3
n=5
I/Ic
on
tro
l
basal ITRPM8
at room temperature (23°C)
+ desflurane 0.20 mM
1 nA
100 ms
basal ITRPM8
at room temperature (23°C)
1.5 nA
100 ms
+ isoflurane 0.20 mM
E
A
D
1.5 nA
+ sevoflurane 0.20 mM
basal ITRPM8
at room temperature (23°C)
100 ms
B C
F
Figure 2.
G
Isoflurane
basal isoflurane
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
no
rma
lize
d N
Po
pe
n
**
5 pA
5 sec
H
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
0 60 120 180 240
0
2000
4000
6000
8000
10000
12000
basal ITRPM8
at 23°C
sevoflurane 0.2 mM
Cu
rre
nt,
pA
Time, s
0 60 120 180 240 3000
500
1000
1500
2000
desflurane 0.2 mM
basal ITRPM8
at 23°C
Time, s
0 60 120 180 240 300 360
0
2000
4000
6000
8000
10000
isoflurane 0.2 mM
basal ITRPM8
at 23°C
Cu
rren
t, p
A
Time, s0 60 120 180 240 300 360 420
0
2000
4000
6000
8000
10000basal I
TRPM8 at 23°C
halothane 0.2 mM
Curr
ent, p
A
Time, s
0 120 240 360 480 600 7200,5
0,6
0,7
0,8
0,9
1,0
isoflurane (0.2mM)
33°C33°C
[Ca
2+] c
, n
M
Time, s
37°C
0
25
50
75
100
*
*
*
*
0.2mMse
voflu
rane
desflu
rane
isof
lura
ne
halo
than
e
CHCl3
CTL
resid
ua
l cu
rren
t, %
*
0 120 240 360 480 600 7200,5
0,6
0,7
0,8
0,9
1,0
[Ca
2+] c
, n
M
Time, s
37°C
33°C
33°C 33°C
A B C
D E F
G
Figure 3.
0
50
100
150
Incubation with CHCl3
(0.25 mM ; 15 min)
I TR
PM
8 a
t 2
3°C
, %
of
co
ntr
ol
CTL
*
H
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
C B A
Figure 4.
basal ITRPM8 at 23 °C
basal ITRPM8 at 23 °C basal ITRPM8 at 23 °C
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
B A
C D
Figure 5.
basal ITRPM8 at 23 °C
0 200 400 600 800
0
500
1000
1500
2000
2500 basal ITRPM8
at 23°C + Poly-L
1800
Cu
rre
nt,
pA
Time, s
halothane 0.5 mM
16000 240 480 720 960 1200 1440
300
600
900
1200basal I
TRPM8 at 23 °C + Poly-L
CHCl3 2.5 mM
Cu
rre
nt,
pA
Time, s
0 100 200 300 400 500 600 700
0
1000
2000
3000
4000
Cu
rre
nt,
pA
Time, s
CHCl3 2.5 mM
+ CPZ 1 µM
CHCl3 2.5 mM
basal ITRPM8
at 23 °C
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
A B
C D
Figure 6.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Figure 7.
A
G F E
B C D *
0 60 120 180 240 300 360 420 480 540 600
0
100
200
300
400
Time, s
Curr
ent density, pA
/pF halothane
BCTC
1
2
3
4
HC 030031
100 ms
200
0 p
A
-600
+100
-100
mV
1
2
3
4
H
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Figure 8.
A B
33
34
35
36
37
38
20 min
***
***
KO-TRPM8KO-TRPM8 WT
Bo
dy t
em
pe
ratu
re,
°C
WT
10 minisolflurane anesthesia
60
80
100
120
140
isolflurane anesthesiaR
esp
ira
tory
fre
qu
en
cy
20 min
N.S
***
KO-TRPM8KO-TRPM8 WTWT
10 min
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t