See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/6316062

Characterisation of TRPM8 as a pharmacophore receptor

Article  in  Cell Calcium · January 2008

DOI: 10.1016/j.ceca.2007.03.005 · Source: PubMed

CITATIONS READS

111 677

3 authors, including:

Ulrich Wissenbach Veit Flockerzi

Universität des Saarlandes Universität des Saarlandes
99 PUBLICATIONS   5,967 CITATIONS    275 PUBLICATIONS   18,786 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Ion channels View project

All content following this page was uploaded by Ulrich Wissenbach on 08 November 2018.

The user has requested enhancement of the downloaded file.

 Cell Calcium 42 (2007) 618–628

Characterisation of TRPM8 as a pharmacophore receptor

Matthias Bödding ∗ , Ulrich Wissenbach, Veit Flockerzi
Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes,
D-66421 Homburg, Germany

Received 12 June 2006; received in revised form 15 March 2007; accepted 21 March 2007
Available online 22 May 2007

Abstract
Some proteins of the transient receptor potential (TRP) family form temperature sensitive ion channels. One member of the melastatin (M)
group, namely TRPM8 is activated by cold and cooling compounds such as menthol and icilin, and its gene is up-regulated in prostate cancer
and other malignancies. Here we characterise the effects of the carboxamides WS-12, CPS-113, CPS-369, the carboxylic acid WS-30 and
the phosphine oxide WS-148 by Ca2+ imaging experiments and whole-cell patch-clamp recordings on TRPM8 expressing human embryonic
kidney (HEK), lymph node prostate cancer (LNCaP) and dorsal root ganglia (DRG) cells. The cooling compounds introduced in this study,
show a dose-dependent and reversible activation of TRPM8 with EC50 values in the nM to low ␮M range. The carboxamide WS-12 is most
potent in activating TRPM8. It is selective, since other TRP proteins are not stimulated at ␮M concentrations and its efficacy with respect to
TRPM8 is similar to the one of icilin. In summary, the compounds described in this study represent new tools to dissect TRPM8 functions and
may serve as chemical leads for the development of additional TRPM8 agonists and novel antagonists. Such compounds may be beneficial
for preventing noxious cold perception. They could also be useful in diagnosis and treatment of most common cancers in which the TRPM8
gene is up-regulated in comparison to the corresponding normal tissue.
© 2007 Elsevier Ltd. All rights reserved.

Keywords: Transient receptor potential (TRP) channels; TRP melastatin type 8 (TRPM8) channels; Cooling compounds; Menthol; Icilin; Cancer; Membrane
targets; Radiotherapy; Calcium imaging; Patch-clamp

1. Introduction (TRPV3 [3–5]), 43 ◦ C (TRPV1 [6]), 52 ◦ C (TRPV2 [7]).

The only known temperature sensors in the nerve end-
ings of mammals belong to the transient receptor potential
(TRP) superfamily of cation channels. Until now, seven
temperature-sensitive TRP channels have been described.
Four members of the vanilloid subfamily are activated by
increases of temperature above 25 ◦ C (TRPV4 [1,2]), 31 ◦ C
Abbreviations: [Ca2+ ]I , intracellular free Ca2+ concentration; DRG,
dorsal root ganglia; GABA, ␥-aminobutyric acid; HEK, human embryonic
kidney; LNCaP, lymph node cancer prostate; PET, positron-emission-
tomography; TRP, transient receptor potential; TRPM, transient receptor
potential melastatin; TRPM3␣2, transient receptor potential melastatin type
3 splice variant ␣2; TRPM8, transient receptor potential melastatin type 8;
TRPV6, transient receptor potential vanilloid type 6
∗ Corresponding author. Tel.: +49 6841 1626242; fax: +49 6841 1626402.
E-mail address: matthias.boedding@uniklinik-saarland.de
(M. Bödding).
TRPM4 and TRPM5, two members of the melastatin sub-
family, are activated by temperatures in the range from 15 to
35 ◦ C [8]. TRPM8 is stimulated by temperatures below 28 ◦ C
[9,10] whereas TRPA1 (ANKTM1) may be sensitive to cold
(<18 ◦ C [11–14]).
The trp-p8 or TRPM8 cDNA was initially identified and
cloned by screening for mRNAs up-regulated in prostate can-
cer [15]. It is also expressed in a number of non prostatic
primary tumors of breast, colon, lung and skin origin whereas
transcripts encoding TRPM8 were hardly detected or not
detected in the corresponding normal human tissues ([15];
reviewed in [16,17]). Using a different approach to identify
and to isolate cDNA which encode proteins sensitive to cool-
ing compounds like menthol and icilin, McKemy et al. [9]
and Peier et al. [10] cloned the TRPM8 cDNA using RNA
from trigeminal neurons. They also showed that TRPM8 is a
Ca2+ permeable cation channel.

0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2007.03.005
 M. Bödding et al. / Cell Calcium 42 (2007) 618–628
In this study, we describe the action of prototype cooling
agents of various chemical structures on TRPM8 channel
activity. The data presented suggest that all compounds used
act as TRPM8 agonists with the carboxamide WS-12 exerting
the highest potency of all known agonists.
2. Materials and methods
2.1. Cell culture, transfection and stable cell lines
Human embryonic kidney (HEK-293) cells were from
the American Type Culture Collection (1573-CRL, ATCC,
Manassas, USA) and cultured as previously described [18].
HEK cells were transiently transfected with 3 ␮g DNA in
9 ␮l of the PolyFect® reagents (Qiagen, Hilden, Germany)
for the experiments shown in Fig. 5. The bicistronic expres-
sion plasmid pdiCaT-Lb contained the entire protein-coding
regions of the human TRPM8 (GeneBankTM , accession
number AY090109, [15]) followed by an internal riboso-
mal entry side and the green fluorescence protein DNA.
Experiments were performed one day after transfection. In
addition cell lines were used which stably express human
TRPM8, human TRPV6b (GeneBankTM , accession num-
ber CAC20417, [19]) or mouse TRPM3␣2 (GeneBankTM ,
accession number AJ544534, [20]). To obtain the TRPM8
cDNA oligonucleotide primers flanking the open reading
frame of the human TRPM8 [15] were used to amplify the
TRPM8 cDNA from prostate cancer by RT-PCR. The cDNA
was subsequently cloned into pcDNA3 (Invitrogen, Karl-
sruhe, Germany). For generation of clones stably expressing
TRPM8, HEK cells transfected with TRPM8-pcDNA3 were
kept in 500 ␮g/ml G418 (Invitrogen). A month later single
surviving cells were isolated by cell sorting using a MoFlo
fluorescent associated cell sorter (DakoCytomation, Ham-
burg, Germany). Cell clones were expanded and tested for
the presence of TRPM8 by immunoblot using affinity purified
polyclonal anti-TRPM8-antiserum 797, generated by immu-
nizing rabbits with a C-terminal peptide derived from human
TRPM8 (Fig. 1).
The human lymph node prostate adenocarcinoma cell line
(LNCaP) was purchased from the American Type Culture
Collection (CRL-10995, Lot 1216242, ATCC, Manassas,
USA) and the Deutsche Sammlung von Mikroorganismen
und Zellkulturen (ACC256, DSMZ, Braunschweig, Ger-
many). Cell culture was as described in [21].
Dorsal root ganglia (DRG) neurons were dissociated and
cultured essentially as previously reported [22]. Adult mice
(C57Bl/6) were anesthetized by the intraperitoneal appli-
cation of a 25% urethane solution (6 ␮l/g). DRG were
quickly removed and incubated in trypsin type 1 (1 mg/ml
medium) and collagenase type II (4 mg/ml medium) for
30 min. Cells were resuspended in Dulbeco’s modified
Eagle’s medium (Invitrogen) supplemented by 10% fetal
calf serum (Invitrogen) and 1% antibiotics (10,000 U/ml
penicillin and 10 mg/ml streptomycin, Invitrogen). Neurones
619
Fig. 1. Stable expression of human TRPM8 in HEK-293 cells. Lysate pro-
teins from HEK cells (lane 1) and the clonal HEK cell lines M8A3 (lane
2) and M8F3 (lane 3), which stably express the human TRPM8, were sep-
arated on a reducing 4, 6.5% SDS-PAGE. Two protein variants of TRPM8
of 124 and 112 kDa were detected with the affinity purified polyclonal anti-
TRPM8-antiserum 797 in the M8A3 and M8F3 cell lines but not in the
parental non transfected HEK cell line. The slower running protein repre-
sents a glycosylated variant of TRPM8 [32]; both variants are not expressed
in the non transfected parental HEK cells. In this study we used the clonal
cell line M8A3 where indicated.
were plated onto poly-l-lysine coated glass coverslips and
kept at 37 ◦ C in a 5% CO2 atmosphere.
2.2. Microfluorimetry
Fluorescent measurements of the intracellular Ca2+ con-
centration ([Ca2+ ]i ) in single cells were carried out at room
temperature essentially as described before [21]. In brief,
cells were incubated in medium containing 2 ␮M fura-2
AM (Molecular Probes, Eugene, OR, USA) at 37 ◦ C for
20 min in the dark and then washed at least three times
with the standard external solution (in mM): 145 NaCl, 2
CaCl2 , 2.8 KCl, 2 MgCl2 , 11 glucose, 10 HEPES, adjusted
to pH 7.2 with NaOH. The Ca2+ imaging set-up consisted of
an inverted microscope (Axiovert S100, Zeiss, Oberkochen,
Germany) with an oil immersion objective (Fluar, ×40/1.3,
Zeiss), a monochromator (Polychrom II, T.I.L.L. Phototon-
ics, Planegg, Germany), and a CCD camera (SensioCam,
PCO, Kelheim, Germany). The Ca2+ indicator dye was
excited alternately at 340 and 380 nm (10 ms exposures) and
images were taken once every second at an emission wave-
length of 510 nm. Fluorescence ratios are plotted versus time
to indicate changes of the intracellular free Ca2+ concentra-
tion ([Ca2+ ]i ). Fura-2 binds Ca2+ with a KD of around 200 nM
[23] which does not allow accurate measurements in the ␮M
range. The drug induced maximal fluorescent increase was,
therefore, not used to determine drug efficacy. Calcium imag-
ing experiments were usually performed at room temperature
(20–23 ◦ C). Recordings with 10 ␮M WS-12 were also done
at body temperature by using a heating stage (TRZ 3700, Carl
Zeiss, Jena, Germany).
620 M. Bödding et al. / Cell Calcium 42 (2007) 618–628

2.3. Electrophysiological recordings and solutions

Membrane currents were recorded in the whole-cell con-

figuration using an EPC-9 amplifier (HEKA Elektronik,
Lambrecht, Germany) as descrybed previously [18]. Patch
pipettes pulled from borosilicate glass (Kimax® ) had resis-
tances between 2 and 3 M when filled with the standard
internal solution (in mM): 145 Cs-glutamate, 10 HEPES, 8
NaCl, 1 MgCl2 , 2 Mg-ATP, 0.1 mM EGTA, adjusted to pH
7.2 with CsOH. The EGTA concentration was 10 mM for
the experiments shown in Fig. 7. Extracellular solution con-
tained (in mM): 145 NaCl, 2 CaCl2 , 2.8 KCl, 2 MgCl2 , 11
glucose, 10 HEPES, adjusted to pH 7.2 with NaOH. Drugs
were applied in the bath solution by a custom-made local
perfusion system. The series resistance was compensated
for 90% and ranged between 3 and 10 M. RS was below
5 M in the experiments shown in Fig. 7. Currents were fil-
tered using an 8-pole Bessel filter at 2.9 kHz and digitised
at 100 ␮s. Voltage ramps (−110 to 90 mV in 50 ms) were
applied at 0.5 Hz from a holding potential of −10 mV using
PULSE software (HEKA Electronics). Several parameters
such as capacitance, series resistance and holding current
were monitored simultaneously at a slower rate (2 Hz) using
the X-Chart display (HEKA Electronics). A liquid junction
potential of 10 mV was applied to all voltages. All exper-
iments were carried out at room temperature (20–23 ◦ C).
Internal solutions were kept on ice to minimise hydrolysis of
ATP.

2.4. Data analysis

Currents were elicited by voltage ramps, measured

at −80 mV and 80 mV, and normalised by dividing the
amplitude by the cell capacitance. Activation curves were
determined from either steady state currents or tail cur-
rents using the voltage protocol shown in Fig. 7. Steady
state currents were measured at the end of each voltage
step as means during a time interval of 5 ms. The conduc-
tance was calculated from these data and divided by the
maximal steady state conductance, which was obtained at
strongly depolarised potentials in the presence of either men-
thol or WS-12. Tail currents were measured 0.5 ms after
repolarisation to 50 mV to avoid contamination by capac-
itative transients. Mean data were calculated by averaging
the tail currents during an interval of 0.5 ms. These val-
ues were divided by the average of the maximal tail current
amplitude in the presence of the agonist. Analysis was per-
formed with PulseFit (HEKA Elektronik) and IGOR (Wave
Metrics, Lake Oswego, OR, USA). Curve fitting was done
with Origin using a sigmoid formulation (Northampton,
MA 01060, USA). Hill coefficients were 0.8 (Fig. 3B), 6.7
(Fig. 3D), 2.3 (Fig. 3F), 1.5 (Fig. 4A), 4.1 (Fig. 4B), 3.9
(Fig. 4C), 2.6 (Fig. 4D) and 3.6 (Fig. 6D). Throughout,
average data are given as mean ± S.E.M. for n number of
cells.
Fig. 2. Chemical structures of menthol, icilin, WS-12, WS-148, WS-30,
CPS-369 and CPS-113. The chemical structures of menthol and icilin
are shown in comparison to menthol-like cooling agents, which form
a chemical class different from what has been reported for TRPM8
agonists. These compounds are the two carboxamides WS-12 (2-Isopropyl-
5-methyl-cyclohexanecarboxylic acid (4-methoxy-phenyl)-amide), CPS-
369 (ethyl-((2-isopropyl-5-methyl-cyclohexanecarboxylic acid)-amino)-
propanoate) and CPS-113 (2-isopropyl-5-methyl-cyclohexanecarboxylic
acid (4-fluoro-phenyl)-amide), the carboxylic acid WS-30 (2-isopropyl-
5-methyl-cyclohexanecarboxylic acid 2,3-dihydroxy-propyl ester) and the
phosphine oxide WS-148 (1-(di-sec-butyl-phosphinoyl)-heptane).
2.5. Chemicals and drugs
WS-12, WS-30 and WS-148 (Fig. 2) were obtained
from Wilkinson Sword in 1985 and synthesized according
to methods referenced in [24]. 2-(1R,2S,5R)-Isopropyl-5-
methyl-cyclohexanecarboxylic acid (4-fluoro-phenyl)-amide
(CPS-113) was synthesized in the laboratory of Dr. Ahamin-
dra Jain, College of Chemistry at the University of
California at Berkeley. (R)-2-[((1R,2S,5R)-2-isopropyl-5-
methyl-cyclohexanecarbonyl)-amino]-propionic acid ethyl
 M. Bödding et al. / Cell Calcium 42 (2007) 618–628 621

ester (CPS-369) was synthesized by Dr. Sergey V. Burov of
the Institute of Macromolecular Chemistry, St. Petersburg,
Russia. Icilin [25] was obtained from Phoenix Pharma-
ceuticals, Belmont, California, USA. Icilin, WS-12 and
CPS-369 were dissolved in DMSO, WS-30, WS-148 in the
external solution and (−)-menthol ((1R,2S,5R)-2-isopropyl-
5-methylcyclohexanol), CPS-113 in 70% ethanol. The term
“menthol” used in this paper refers to this isomer. These
stock solutions were diluted to the final drug concentration
by adding the appropriate volume of Ringer’s solution. All
reagents were from Sigma (Deisenhofen, Germany) except
Fura-2 AM (Molecular Probes) and collagenase type II
(Biochrom, Berlin, Germany).
3. Results
3.1. Menthol and icilin induced Ca2+ signals in TRPM8
expressing HEK cells
TRPM8 is a Ca2+ permeable channel [9,10]. Thus, it is
possible to monitor its activity in Ca2+ imaging experiments

Fig. 3. Agonist induced cytosolic Ca2+ signals in TRPM8 expressing HEK cells. Intracellular Ca2+ measurements were performed from single HEK cells stably
expressing the TRPM8 protein. The extracellular Ringer’s solution contained 2 mM Ca2+ . The bar indicates the time during which the agonist was applied onto
the cells. Ca2+ imaging was performed as described under Section 2. (A) Fluorescent changes were evoked by menthol using 300 nM (yellow trace), 1 ␮M
(orange), 3 ␮M (red), 10 ␮M (bright green), 30 ␮M (dark green), 100 ␮M (blue), 300 ␮M (black). Averaged data are shown. (B) Concentration–response curve
of the experiments in (A). The menthol-induced maximal fluorescent increase was determined for each concentration (n = 37 for 300 nM, n = 90 for 1 ␮M, n = 97
for 3 ␮M, n = 136 for 10 ␮M, n = 72 for 30 ␮M, n = 101 for 100 ␮M, n = 61 for 300 ␮M, n = 45 for 1 mM). (C) Example for the icilin-induced fluorescent changes
at a concentration of 2 ␮M. (D) Concentration–response curve for icilin (n = 50 for 100 nM, n = 118 for 300 nM, n = 48 for 1 ␮M, n = 27 for 2 ␮M, n = 27 for
3 ␮M, n = 18 for 10 ␮M). (E) Representative recording with 300 nM WS-12. (F) Concentration–response curve for WS-12 (n = 41 for 10 nM, n = 12 for 30 nM,
n = 23 for 50 nM, n = 143 for 100 nM, n = 59 for 300 nM, n = 40 for 1 ␮M, n = 39 for 10 ␮M). Data are shown as means ± S.E.M. in the concentration–response
curves in (B), (D) and (F).
622 M. Bödding et al. / Cell Calcium 42 (2007) 618–628

upon application of cooling compounds such as menthol and
icilin [26]. The extracellular application of menthol leads to a
dose-dependent increase of the fura-2 fluorescence ratio indi-
cating an elevation of the free cytosolic Ca2+ concentration
(Fig. 3A). Increasing the menthol concentration accelerated
the initial Ca2+ upstroke. The EC50 was 10.4 ␮M (Fig. 3B)
which is similar to the values reported previously [9,27,28].
Icilin which is more potent than menthol [9,27,28] increases
the [Ca2+ ]i with an EC50 of 1.4 ␮M icilin. This value is almost
one order of magnitude lower than that of menthol (Fig. 3D).
Furthermore, the concentration-response curve was steeper
for icilin in comparison to the one for menthol (Fig. 3D).
Another difference was the transient behaviour of the icilin-
induced Ca2+ elevation in comparison to that elicited by
menthol (Fig. 3C). There were no significant fluorescent
changes if both drugs were applied in the absence of exter-
nal Ca2+ (data not shown). Thus, Ca2+ influx and not Ca2+
release is responsible for the menthol and icilin induced Ca2+

Fig. 4. Concentration–response curves for TRPM8 agonists. Experiments were performed as described for Fig. 3. (A) WS-148 was applied in the standard
external solution using 100 nM (n = 34), 250 nM (n = 30), 1 ␮M (n = 40), 5 ␮M (n = 53), 10 ␮M (n = 45) and 100 ␮M (n = 33). (B) WS-30 was tested in the
following concentrations: 1 ␮M (n = 32), 3 ␮M (n = 36), 5 ␮M (n = 37), 10 ␮M (n = 37), 30 ␮M (n = 31) and 100 ␮M (n = 42). (C) CPS-369 was applied at
300 nM (n = 33), 1 ␮M (n = 50), 3 ␮M (n = 50), 10 ␮M (n = 59) and 30 ␮M (n = 86). (D) CPS-113 was used at 100 nM (n = 47), 300 nM (n = 46), 1 ␮M (n = 59),
3 ␮M (n = 46), 10 ␮M (n = 57). (E) Normalised concentration response curves for TRPM8 agonists. Fluorescent ratios were divided by the maximal fluorescent
increase and subtracted by the fluorescent ratio at the lowest drug concentration. Data were taken from Fig. 3B, D, F and Fig. 4A–D. The order of apparent EC50
values was: WS-12 (193 nM) < CPS-113 (1.2 ␮M) < icilin (1.4 ␮M) < CPS-369 (3.6 ␮M CPS-369) < WS-148 (4.1 ␮M) < WS-30 (5.6 ␮M) < menthol (10.4 ␮M).
Mean data are shown without S.E.M. for the sake of clarity.
 M. Bödding et al. / Cell Calcium 42 (2007) 618–628 623

signals. No Ca2+ elevations were seen if the bath solution was
applied with the solvent without menthol or icilin (n = 61,
data not shown). Similarly, no change in the fluorescent signal
was detectable by applying the compounds to non-transfected
HEK cells (data not shown). Taken together, these results are
in good agreement to previously published results [9,27,28]
and demonstrate that menthol and icilin stimulate TRPM8
mediated Ca2+ influx in the cells used for this study.
3.2. WS-12, WS-148, WS-30, CPS-369 and CPS-113
induced Ca2+ signals in TRPM8 expressing HEK cells
The following cooling compounds, whose chemical
structures are shown in Fig. 2, were studied on TRPM8
expressing HEK cells. All substances increased the [Ca2+ ]i
of TRPM8-expressing HEK cells in a dose-dependent way
(Figs. 3 and 4). WS-12 was the most potent drug tested
(Fig. 3E and F). Its EC50 of 193 nM was almost one order
of magnitude below the value of icilin. The EC50 of the other
compounds were 4.1 ␮M for WS-148, 5.6 ␮M for WS-30,
3.6 ␮M for CPS-369 and 1.2 ␮M for CPS-113 (Fig. 4). For
all substances the same control experiments were performed
as for menthol and icilin, showing no change in the [Ca2+ ]i . In
addition, it was tested whether the WS-12 response was addi-
tive to the ones of known TRPM8 agonists. When cells were
challenged with WS-12, the consecutive icilin induced rise
in the [Ca2+ ]i was abolished (data not shown) indicating that
both substances act at the same molecular target. It is unlikely,
that the WS-12 induced increase in the [Ca2+ ]i is responsible
for the missing icilin induced Ca2+ rise since icilin requires
high [Ca2+ ]i to achieve full efficacy [27]. Taken together the
results suggest that WS-12, WS-148, WS-30, CPS-369 and
CPS-113 act as agonists on TRPM8 channel activity.
TRPM8 activates already at temperatures around 28 ◦ C
[9,10]. As a consequence the [Ca2+ ]i is increased at room
temperature in comparison to body temperature (data not
shown). To test whether WS-12 also activates TRPM8 under
more physiological conditions, additional experiments were
performed at 37 ◦ C. A high concentration of WS-12 (10 ␮M)
induced similar cytosolic Ca2+ elevations at 37 ◦ C and 22 ◦ C
(data not shown, n = 50 at 37 ◦ C and n = 23 at 22 ◦ C). It is,
therefore, possible to use WS-12 as a TRPM8 agonist at body
temperature.
The selectivity of these drugs was investigated by studying
their effects on other TRP proteins such as the Ca2+ channel
TRPV6 and cation channel TRPM3. Functional expression of
these proteins in HEK cells was confirmed by Western blot
analysis, Ca2+ imaging experiments and electrophysiologi-
cal recordings in this study as previously shown [20,21]. No
change in the [Ca2+ ]i was detected on cells stably express-
ing TRPV6 after applying WS-12 (n = 82), WS-148 (n = 61),
WS-30 (n = 43) and CPS-113 (n = 41) at a high concentration
of 100 ␮M. Additional control experiments were performed
with WS-12 on transiently transfected cells. No differences
were detectable between HEK cells transiently transfected
with TRPV6 (n = 5 for 10 ␮M WS-12 and n = 11 for 20 ␮M
WS-12) and non transfected cells (n = 24 for 10 ␮M WS-
12 and n = 13 for 20 ␮M WS-12). Similarly, 100 ␮M WS-12
(n = 44), WS-30 (n = 23), WS-148 (n = 30) and CPS-113
(n = 25) did not affect the [Ca2+ ]i of the TRPM3␣2 express-

Fig. 5. WS-12 effects on HEK cells transiently transfected with the TRPM8 cDNA. (A) Time-course of fluorescent changes due to WS-12. The agonist
concentration was 2 nM (n = 29), 100 nM (n = 29), and 20 ␮M (n = 13). The insert shows the control recording from non transfected HEK cells (n = 76 for 2 nM,
n = 50 for 100 nM, and n = 22 for 20 ␮M). Mean data are presented. (B) Current–voltage (I–V)-relationship of HEK cells transiently expressing TRPM8. Voltage
steps were applied as shown in the inset and described in Section 2. Currents were measured at the end of each pulse. Mean current densities are plotted vs.
membrane potential (n = 5). (C) A typical I–V-curve for a transiently transfected cell is shown (n = 15). The current trace was recorded in response to a voltage
ramp of 50 ms duration that ranged from −110 mV to 90 mV. The dashed line represents zero current.
624 M. Bödding et al. / Cell Calcium 42 (2007) 618–628

ing HEK cell line indicating that these compounds can be 3.4. Icilin and WS-12 induced outward currents in
used to discriminate among TRP channels. TRPM8 expressing HEK cells

3.3. WS-12 induced Ca2+ signals in transiently
transfected HEK-TRPM8 cells
The effect of WS-12 on TRPM8 was further investigated
using transiently transfected HEK cells. Functional expres-
sion of TRPM8 in these cells was verified by patch-clamp
recordings in the whole-cell configuration. Large outward
currents with a reversal potential close to 0 mV were recorded
in the whole-cell mode by applying either voltage steps
or ramps (Fig. 5B and C). Similar current–voltage (I–V)-
relationships, though smaller current-densities, were mea-
sured from cells stably expressing TRPM8 (Fig. 6). Again,
WS-12-induced elevations of the [Ca2+ ]i (Fig. 5A) very sim-
ilar as it does in the cells stably expressing TRPM8 (Fig. 3E).
To compare the effects of WS-12 with those of icilin
in more detail patch-clamp experiments were performed in
the whole-cell mode. Icilin (5 ␮M) reversibly activated an
outwardly rectifying current which reverses around 0 mV
(Fig. 6A). The voltage dependence of this cation cur-
rent resembles that of TRPM8, shown in Fig. 5B and C.
A very similar outward current developed if cells were
challenged with the same concentration of WS-12 indicat-
ing activation of TRPM8 (Fig. 6B). A typical recording
in which WS-12 even at such a low concentration of
100 nM induced the reversible current increase is shown in
Fig. 6C. The concentration dependence was analysed by
measuring the WS-12 induced current increase at 80 mV
(Fig. 6D). The calculated apparent EC50 of 680 nM is

Fig. 6. Icilin and WS-12 induced currents in TRPM8 expressing HEK cells. Whole-cell patch-clamp recordings were carried out from HEK cells stably
expressing the TRPM8 protein. (A) Icilin (5 ␮M) was applied in the external solution. Current densities at −80 and 80 mV are plotted vs. time. Representative
I–V curves from the time points indicated are shown. (B, C) WS-12 was used at a concentration of 5 ␮M or 100 nM, respectively. (D) Concentration–response
curve for WS-12. The increase of current–densities as means with double-sided S.E.M. are shown in dependence of the WS-12 concentration (n = 8 for 100 nM,
n = 9 for 300 nM, n = 9 for 1 ␮M, n = 7 for 3 ␮M, n = 11 for 10 ␮M). (E) Agonist (10 ␮M) induced increase of the inward and outward current measured at −80
and 80 mV. Current densities at both potentials were not statistically different if measured before the application of either icilin or WS-12. Averaged data with
one-sided S.E.M. are shown in the histogram (n = 6 for icilin and n = 11 for WS-12).
 M. Bödding et al. / Cell Calcium 42 (2007) 618–628
Fig. 6. (Continued ).
slightly higher than the one determined in the Ca2+ imag-
ing experiments (EC50 ≈ 193 nM; Fig. 3F) which is not
unexpected since the experimental design is completely dif-
ferent. The efficacy of WS-12 was compared to one of the
icilin by applying saturating concentrations of both drugs
(10 ␮M). The current increase was not statistically differ-
ent (Fig. 6E) suggesting similar maximal effects of both
compounds.
3.5. WS-12 activated TRPM8 by shifting the
voltage-dependence of activation
It has been shown that menthol shifts the activation curve
of TRPM8 towards negative potentials which results in acti-
vation of TRPM8 at room temperature [29,30]. Likewise,
cooling shifts the voltage-dependence of TRPM8 towards
physiologically relevant potentials. WS-12 induces a cooling
sensation similar to menthol [24] and WS-12, like menthol,
activated TRPM8 by causing a leftward shift of the voltage-
dependence of activation (Fig. 7).
625
3.6. Effect of WS-12 on cells endogenously expressing
TRPM8
Can WS-12 be used to study endogenous TRPM8
channels? Lymph node prostate cancer (LNCaP) cells are
supposed to express the TRPM8 mRNA and protein [31].
However, the application of WS-12 (n = 219 for 10 ␮M and
n = 28 for 50 ␮M) was without effect in Ca2+ imaging experi-
ments (Fig. 8A). This was also the case for icilin (Fig. 8A) and
menthol (data not shown, n = 50 for 1 mM). These findings
were confirmed by using LNCaP cells from a second, inde-
pendent source. One possible explanation for these results
is lack of functional expression of TRPM8 in LNCaP cells.
Maybe, LNCaP cells do express truncated variants which do
not encode functional TRPM8 channels. Indeed, we did iden-
tify such TRPM8 variants when we cloned the TRPM8 cDNA
[30,32] from poly A+ RNA isolated from human prostate
cancer (reviewed in [33]).
Dorsal root ganglia neurons are known to express TRPM8
mRNA [5,9]. These primary cells were used to study the
effect of WS-12 on TRPM8 under physiological conditions.
626 M. Bödding et al. / Cell Calcium 42 (2007) 618–628

Fig. 7. Menthol and WS-12 induced currents in TRPM8 expressing HEK cells. (A) Current traces in response to the indicated voltage protocol. Typical
recordings are shown before and during application of either menthol (1 mM) or WS-12 (10 ␮M). (B) Tail currents were measured during the repolarisation
step to 50 mV and divided by the maximal tail current amplitude from the traces in (A). Representative data are shown in the absence (n = 19) or presence of
either menthol (n = 11) or WS-12 (n = 8). (C) The fraction of open channels was determined from steady-state currents at the end of the voltage steps. The
voltage relations of the normalised conductances are shown as G/Gmax where Gmax represents the maximal steady-state conductance (n = 8).

Approximately 45% of the selected DRG neurones under 4. Discussion

investigation respond to menthol (1 mM) with an increase of
the [Ca2+ ]i (230 of 509 cells). WS-12 induced in about 3% of
the cells a Ca2+ elevation (Fig. 8B, 17 of 509 cells). Raising
the WS-12 concentration (50 ␮M) resulted in an increase of
responding cells to 33% (35 of 106 cells). Thus, WS-12 is a
useful tool to study TRPM8 in its native environment, though
the potency is lower than in the experiments on transfected
cells.
TRPM8 is a cation channel which is activated by low tem-
perature and cooling compounds [9,27]. Naturally occurring
monoterpens such as menthol and the synthetic super-cooling
agent icilin [25] are commonly used to activate and study
TRPM8 channel activity. Here, chemicals with diverse struc-
tures are shown to act as TRPM8 agonists. The carboxamides
(WS-12, CPS-369, CPS-113), the carboxylic acid ester (WS-
 M. Bödding et al. / Cell Calcium 42 (2007) 618–628
Fig. 8. Effect of WS-12 on LNCaP cells and DRG neurones. Time-course of
fluorescent changes are shown for LNCaP and DRG cells. (A) Application
of WS-12 (10 ␮M), icilin (10 ␮M) and ionomycin (5 ␮M) to LNCaP cells
(n = 219) are represented by the upper bar. (B) DRG cells were stimulated
with WS-12 (10 ␮M) and only 17 of 509 neurones respond with a fluorescent
increase. Mean data from these 17 cells are shown with double-sided S.E.M.
30) and the phosphine oxide (WS-148) all activated TRPM8
channels with EC50 in nM to low ␮M range. These com-
pounds activate TRPM8 but not related TRP channels like
TRPM3 and TRPV6. The potency of WS-12 in stimulat-
ing TRPM8 at concentrations lower than icilin is remarkable
and has not been described previously. Like menthol, WS-12
seems to activate TRPM8 mediated cation currents by shift-
ing the voltage dependence of the activation curves to the left
toward more physiological membrane potentials [29,30].
The TRPM8 agonists, especially WS-12, are new tools
to study TRP channels. Chemical modification of WS-12,
for example, with replacement of the para-methoxy by fluo-
rine results in the analog CPS-113 with significant activity on
TRPM8. Apparently both compounds may function as chem-
ical leads for synthesising even more potent TRPM8 agonists.
They also might be useful for developing TRPM8 antagonists
and radioligands, which can be used to characterise binding
sites within the TRPM8 protein.
Beside the advantage of agonists to characterise the
TRPM8 protein and function, these compounds should also
be considered as leads to develop diagnostic tracers and drugs
for patients suffering from most common cancers. TRPM8 is
a promising drug target, since its mRNA is not only expressed
in sensory neurons but also and predominantly in malignant
tissue ([15]; reviewed in [17]). This has especially been stud-
ied for prostate cancer [34,35], one of the most common
627
malignant diseases worldwide [36]. Since TRPM8 forms a
Ca2+ permeable channel, its activation can result in cytoso-
lic Ca2+ elevations. A consequence of a sustained rise in
the [Ca2+ ]i can be DNA transcription, finally leading to cell
proliferation (reviewed in [37,38]). Provided that TRPM8
transcripts are translated into functional plasma membrane
proteins the selective and specific TRPM8 agonists described
in this study might modulate Ca2+ influx and disturb the
cytosolic Ca2+ homeostasis, which could interfere growth
of malignant cells. The incorporation of a radioactive halo-
gene atom such as 18 F in the carboxamide structure of WS-12
leads to CPS-113-[18 F] which could function as a new tool
for radiotherapy and positron-emission-tomography (PET).
In summary these key structures will be helpful in studying
TRPM8 and may be clinically useful in the near future.
Note added in proof
After submission of this paper, similar observations were
reported [39].
Acknowledgements
We thank Prof. Edward T. Wei from the University of
California at Berkeley for the generous gift of all com-
pounds tested in this study. The help of Dr. Stefan Philipp
in the generation of the stable HEK-TRPM8 cell lines by
cell sorting is gratefully acknowledged. We also like to
thank Mrs. Heidi Löhr, Birgit Spohrer and Christine Jung
for excellent technical assistance. This work was supported
by the Wilhelm Sander-Stiftung, the Fonds der Chemischen
Industrie and the Forschungsausschuss der Universität des
Saarlandes.
References
[1] H. Watanabe, J. Vriens, S.H. Suh, C.D. Benham, G. Droogmans, B.
Nilius, Heat-evoked activation of TRPV4 channels in a HEK293 cell
expression system and in native mouse aorta endothelial cells, J. Biol.
Chem. 277 (2002) 47044–47051.
[2] A.D. Güler, H. Lee, T. Iida, I. Shimizu, M. Tominaga, M. Caterina,
Heat-evoked activation of the ion channel, TRPV4, J. Neurosci. 22
(2002) 6408–6414.
[3] H. Xu, I.S. Ramsey, S.A. Kotecha, M.M. Moran, J.A. Chong, D. Law-
son, P. Ge, J. Lilly, I. Silos-Santiago, Y. Xie, P.S. DiStefano, R. Curtis, D.
Clapham, TRPV3 is a calcium-permeable temperature-sensitive cation
channel, Nature 418 (2002) 181–186.
[4] G.D. Smith, M.J. Gunthorpe, R.E. Kelsell, P.D. Hayes, P. Reilly,
P. Facer, J.E. Wright, J.C. Jerman, J.P. Walhin, L. Ooi, J. Egerton,
K.J. Charles, D. Smart, A.D. Randall, P. Anand, J.B. Davis, TRPV3
is a temperature-sensitive vanilloid receptor-like protein, Nature 418
(2002) 186–190.
[5] A.M. Peier, A.J. Reeve, D.A. Andersson, A. Moqrich, T.J. Earley,
A.C. Hegarden, G.M. Story, S. Colley, J.B. Hogenesch, P. McIntyre,
S. Beaven, A. Patapoutian, A heat-sensitive TRP channel expressed in
keratinocytes, Science 296 (2002) 2046–2049.
628 M. Bödding et al. / Cell Calcium 42 (2007) 618–628
[6] M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D.
Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel
in the pain pathway, Nature 389 (1997) 816–824.
[7] M.J. Caterina, T.A. Rosen, M. Tominaga, A.J. Brake, D. Julius, A
capsaicin-receptor homologue with a high threshold for noxious heat,
Nature 398 (1999) 436–441.
[8] K. Talavera, K. Yasumatsu, T. Voets, G. Droogmans, N. Shige-
mura, Y. Ninomiya, R.F. Margolskee, B. Nilius, Heat activation of
TRPM5 underlies thermal sensitivity of sweet taste, Nature 438 (2005)
1022–1025.
[9] D.D. McKemy, W.M. Neuhausser, D. Julius, Identification of a cold
receptor reveals a general role for TRP channels in thermosensation,
Nature 416 (2002) 52–58.
[10] A.M. Peier, A. Moqrich, A.C. Hergarden, A.J. Reeve, D.A. Ander-
sson, G.M. Story, T.J. Earley, I. Dragoni, P. McIntyre, S. Bevan, A.
Patapoutian, A TRP channel that senses cold stimuli and menthol, Cell
108 (2002) 705–715.
[11] G.M. Story, A.M. Peier, A.J. Reeve, S.R. Eid, J. Mosbacher, T.R. Hricik,
T.J. Earley, A.C. Hergarden, D.A. Andersson, S.W. Hwang, P. McIn-
tyre, T. Jegla, S. Bevan, A. Patapoutian, ANKTM1, a TRP-like channel
expressed in nociceptive neurons, is activated by cold temperatures,
Cell 112 (2003) 819–829.
[12] S.E. Jordt, D.M. Bautista, H.H. Chuang, D.D. McKemy, P.M. Zygmunt,
E.D. Hogestatt, I.D. Meng, D. Julius, Mustard oils and cannabinoids
excite sensory nerve fibres through the TRP channel ANKTM1, Nature
427 (2004) 260–265.
[13] D.M. Bautista, S.E. Jordt, T. Nikai, P.R. Tsuruda, A.J. Read, J. Poblete,
E.N. Yamoah, A.I. Basbaum, D. Julius, TRPA1 mediates the inflam-
matory actions of environmental irritants and proalgesic agents, Cell
124 (2006) 1269–1282.
[14] K.Y. Kwan, A.J. Allchorne, M.A. Vollrath, A.P. Christensen, D.S.
Zhang, C.J. Wolf, D.P. Corey, TRPA1 contributes to cold, mechanical,
and chemical nociception but is not essential for hair-cell transduction,
Neuron 50 (2006) 277–289.
[15] L. Tsavaler, M.H. Shapero, S. Morkowski, R. Laus, Trp-p8, a novel
prostate-specific gene, is up-regulated in prostate cancer and other
malignancies and shares high homology with transient receptor poten-
tial calcium channel proteins, Cancer Res. 61 (2001) 3760–3769.
[16] U. Wissenbach, B. Niemeyer, N. Himmerkus, T. Fixemer, H. Bonkhoff,
V. Flockerzi, TRPV6 and prostate cancer: cancer growth beyond the
prostate correlates with increased TRPV6 Ca2+ channel expression,
Biochem. Biophys. Res. Commun. 322 (2004) 1359–1363.
[17] M. Bödding, TRP proteins and cancer, Cell. Signal. 19 (2007) 617–624.
[18] M. Bödding, U. Wissenbach, V. Flockerzi, The recombinant human
TRPV6 channel functions as a Ca2+ sensor in HEK and RBL cells, J.
Biol. Chem. 277 (2002) 36656–36664.
[19] U. Wissenbach, B.A. Niemeyer, T. Fixemer, A. Schneidewind, C. Trost,
A. Cavalie, K. Reus, E. Meese, H. Bonkhoff, V. Flockerzi, Expres-
sion of CaT-like, a novel calcium-selective channel, correlates with the
malignancy of prostate cancer, J. Biol. Chem. 276 (2001) 19461–19468.
[20] J. Oberwinkler, A. Lis, K.M. Giehl, V. Flockerzi, S. Philipp, Alternative
splicing switches the divalent cation selectivity of TRPM3 channels, J.
Biol. Chem. 280 (2005) 22540–22548.
[21] M. Bödding, C. Fecher-Trost, V. Flockerzi, Store-operated Ca2+ cur-
rent and TRPV6 channels in LNCaP cells, J. Biol. Chem. 278 (2003)
50872–50879.
[22] M. Murakami, B. Fleischmann, C. De Felipe, M. Freichel, C. Trost,
A. Ludwig, U. Wissenbach, H. Schwegler, F. Hofmann, J. Hescheler,
View publication stats
V. Flockerzi, A. Cavalié, Pain perception in mice lacking the beta3
subunit of voltage-activated calcium channels, J. Biol. Chem. 277
(2002) 40342–40351.
[23] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+
indicators with greatly improved fluorescence properties, J. Biol. Chem.
260 (1985) 3440–3450.
[24] H.R. Watson, R. Hems, D.G. Rowsell, D.J. Spring, New compounds
with the menthol cooling effect, J. Soc. Cosmet. Chem. 29 (1978)
185–200.
[25] E.T. Wei, D.A.J. Seid, AG-3-5: a chemical producing sensation of cold,
Pharm. Pharmacol. 35 (1983) 110–112.
[26] A. Patapoutian, A.M. Peier, G.M. Story, V. Viswanath, ThermoTRP
channels and beyond: mechanisms of temperature sensation, Nat. Rev.
Neurosci. 4 (2003) 529–539.
[27] H.H. Chuang, W.M. Neuhausser, D. Julius, The super-cooling agent
icilin reveals a mechanism of coincidence detection by a temperature-
sensitive TRP channel, Neuron 43 (2004) 859–869.
[28] H.J. Behrendt, T. Germann, C. Gillen, H. Hatt, R. Jostock, Characteriza-
tion of the mouse cold-menthol receptor TRPM8 and vanilloid receptor
type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay,
Br. J. Pharmacol. 141 (2004) 737–745.
[29] S. Brauchi, P. Orio, R. Latorre, Clues to understanding cold sensa-
tion: thermodynamics and electrophysiological analysis of the cold
receptor TRPM8, Proc. Nat. Acad. Sci. U.S.A. 101 (2004) 15494–
15499.
[30] T. Voets, G. Droogmans, U. Wissenbach, A. Janssens, V. Flockerzi,
B. Nilius, The principle of temperature-dependent gating in cold- and
heat-sensitive TRP channels, Nature 430 (2004) 748–754.
[31] L. Zhang, G.J. Barritt, Evidence that TRPM8 is an androgen-dependent
Ca2+ channel required for the survival of prostate cancer cells, Cancer
Res. 64 (2004) 8365–8373.
[32] I. Erler, D.M. Al-Ansary, U. Wissenbach, T.F. Wagner, V. Flockerzi,
B.A. Niemeyer, Trafficking and assembly of the cold-sensitive TRPM8
channel, J. Biol. Chem. 281 (2006) 38396–38404.
[33] A. Lis, U. Wissenbach, S.E. Philipp, Transcriptional regulation
and processing increase the functional variability of TRPM chan-
nels, Naunyn-Schmiedeberg’s Arch. Pharmacol. 371 (2005) 315–
324.
[34] S. Fuessel, D. Sickert, A. Meye, U. Klenk, U. Schmidt, M. Schmitz,
A.K. Rost, B. Weigle, A. Kiessling, M.P. Wirth, Multiple tumor marker
analyses (PSA, hK2, PSCA, trp-p8) in primary prostate cancers using
quantitative RT-PCR, Int. J. Oncol. 23 (2003) 221–228.
[35] G. Bidaux, M. Roudbaraki, C. Merle, A. Crepin, P. Delcourt, C. Slo-
mianny, S. Thebault, J.L. Bonnal, M. Benahmed, F. Cabon, B. Mauroy,
N. Prevarskaya, Evidence for specific TRPM8 expression in human
prostate secretory epithelial cells: functional androgen receptor require-
ment, Endocr. Relat. Cancer 12 (2005) 367–382.
[36] D.M. Parkin, F.J. Bray, S.S. Devesa, Cancer burden in the year 2000.
The global picture, Eur. J. Cancer 37 (2001) S4–S66.
[37] M.J. Berridge, Calcium signalling and cell proliferation, Bioessays 17
(1995) 491–500.
[38] L. Munaron, S. Antoniotti, A. Fiorio Pla, D. Lovisolo, Blocking Ca2+
entry: a way to control cell proliferation, Curr. Med. Chem. 11 (2004)
1533–1543.
[39] Y.B. Beck, G. Bidaux, A. Bavencoffe, L. Lemonnier, S. Thebault, Y.
Shuba, G. Barrit, R. Skryma, N. Prevarskaya, Prospects for prostate
cancer imaging and therapy using high-affinity TRPM8 activators, Cell
Calcium 41 (2007) 285–294.