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1Departments of Biological Chemistry and Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland 21205; and 2Departments of Oral and Craniofacial Biological Sciences and 3Anatomy and Neurobiology, University of Maryland Dental School, Baltimore, Maryland 21201
Submitted 23 September 2002; accepted in final form 5 March 2003
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ABSTRACT |
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INTRODUCTION |
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;
Peier et al. 2002b;
Smith et al. 2002;
Souslova et al. 2000;
Xu et al. 2002), the molecular
mechanisms underlying the detection of cool and painfully cold stimuli remain
less well understood. Cooling-evoked changes in membrane potential, ionic
conductance, or intracellular calcium can be recapitulated in sensory neurons
cultured from dorsal root (DRG) and trigeminal ganglia (TG)
(McKemy et al. 2002;
Okazawa et al. 2002; Reid and
Flonta
2001a,b,
2002;
Suto and Gotoh 1999; Thut et
al. 2003; Viana et al. 2002).
Data from several recent electrophysiological studies have revealed two
general mechanisms that appear to underlie cold transduction in these neurons.
One mechanism involves the cooling-induced activation of a nonselective
cationic current that is present in approximately 10% of cultured sensory
neurons (Okazawa et al. 2002;
Reid and Flonta 2001a,
2002) and can be potentiated
by the cooling agent menthol (Reid and Flonta
2001a,
2002). A second mechanism
entails the cooling-induced inhibition of certain potassium currents, which
results in net membrane depolarization
(Reid and Flonta 2001b;
Viana et al. 2002). In
addition, a distinct potassium current has been reported to mask
cooling-induced depolarization in a subset of apparently cold-insensitive
sensory neurons (Viana et al.
2002).
Several candidate cold-transducing molecules have been identified based on
their behavior in heterologous expression systems and on their endogenous
expression in sensory neurons. One member of the two-pore potassium channel
family, TREK-1, appears to be inhibited by cooling when expressed in
Xenopus oocytes (Maingret et al.
2000). In contrast, the epithelial sodium channel, ENaC, mediates
a ligand-independent, amiloride-sensitive current that can be stimulated by
cooling (Askwith et al. 2001).
A third candidate cold receptor, TRPM8, is a nonselective cation channel
expressed in a subset of small diameter sensory neurons
(McKemy et al. 2002;
Peier et al. 2002a). In
heterologous expression systems, this channel can be activated by either cold
temperature or menthol (McKemy et al.
2002; Peier et al.
2002a), making it a particularly strong candidate for a cold
transducer.
To better understand the different patterns of cold responses observed in vitro, we used the single-cell reverse transcription-polymerase chain reaction (RT-PCR) method to assay the expression of candidate cold transducing molecules in functionally characterized cultured trigeminal neurons.
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METHODS |
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Adult male Sprague-Dawley rats (150–300 g, Harlan Sprague-Dawley, Indianapolis, IN) were housed in a vivarium under a 12-h light:dark cycle and were fed standard rat diet and water ad libitum prior to use in experiments. Rats were anesthetized with a cocktail of ketamine, xylazine, and acepromazine and then decapitated. All procedures performed on rats were approved by institutional animal care and use committees.
Trigeminal ganglia were removed, dissociated in 0.125% collagenase (Roche,
Indianapolis, IN), triturated in 0.25% trypsin (Worthington, Lakewood, NJ),
and separated on a Percoll (Sigma, St. Louis, MO) gradient
(Eckert et al. 1997). Cells
were plated onto poly-L-ornithine-coated coverslips and incubated
in MEM with vitamins, penicillin/streptomycin, 10% fetal bovine serum, and 20
ng/mL NGF (Invitrogen, Carlsbad, CA) for 1 day (3% CO2, 37°C).
Cells were loaded with 2.5 µM fura-2-acetomethoxy ester (TEF Labs, Austin,
TX) for 20 min at room temperature (approximately 22°C), followed by a
20-min incubation in bath solution (see below) to enable deesterification of
the fura. For 10 min of this second incubation period, cells were stained with
10 µg/ml fluorescein-tagged IB4 (Sigma)
(Stucky and Lewin 1999).
Coverslips were placed in a recording chamber and continuously perfused (2
ml/min) with bath solution containing the following (in mM): 130 NaCl, 3 KCl,
2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 10 glucose (pH 7.4 with
Tris base, adjusted to 325 mOsm with sucrose). Coverslips were held at
34°C and then cooled at a rate of 2.5°C/s to 14°C, or stimulated
with 100 µM menthol for 60 s at 34°C. Prolonged cold stimuli (5 min at
14°C) were applied to a subset of cells, as indicated. Temperature was
controlled by a Cell Micro Control system (Norfolk, VA) consisting of a
Peltier unit and a resistive heater with feedback control and was monitored by
a thermistor placed within 150 µm of the microscopic field. Fluorescent
measurements were made on a Nikon inverted microscope (Image Systems,
Gaithersburg, MD) using a CCD camera (Roper Scientific, Trenton, NJ), filter
wheel (Sutter, Novato CA), and Metafluor software (Universal Imaging, West
Chester, PA). The ratio of fluorescence emission (510 nm) at 340/380 nm
excitation was measured at 1-s intervals. Kinetic fitting of responses for
half-time (t1/2) calculations was performed using Prism
software (Graphpad, San Diego, CA).
Single-cell RT-PCR
Functionally characterized neurons were collected with large-bore
(approximately 30 µm) glass pipettes and expelled into microcentrifuge
tubes containing RT mix (Dulac
1998). RT-PCR was performed as described elsewhere
(Dulac 1998) except that an
anchored primer (5'-ttttttttttttttttttvn-3'; v = a, c, or g; n =
a, c, g, or t) and 50 U Superscript II (Invitrogen) were used for reverse
transcription. For each experiment, negative controls were performed by
omitting reverse transcriptase or using a cell-free bath aspirate as template.
Nine percent of the first-strand cDNA from a given cell was used as template
in a PCR reaction containing 1x Titanium Taq PCR buffer
(Clontech), 0.4 µM outer primers, 0.2 mM dNTPs, and 0.5 µL Titanium
Taq (Clontech); primer sequences are listed in
Table 1. Reactions were
incubated at 94°C for 4 min and then cycled 35 times at 94°C/30 s,
68°C/1 min, before a final extension step of 68°C for 4 min. Four
percent of each initial PCR product served as template in a subsequent PCR
reaction using a nested primer pair. For each PCR experiment, whole TG-derived
cDNA was used as a positive control template. Negative controls included no
input template. Twenty percent of each PCR product was electrophoresed on 2%
agarose–ethidium bromide gels and photographed. Only neurons producing
detectable amplification of a housekeeping gene (cyclophilin) were analyzed
further. For each gene assayed, specificity was confirmed by sequencing the
nested PCR product amplified from whole TG. Unless otherwise indicated, all
quantitative comparisons are presented as mean ± SE and statistical
comparisons were performed using 
2 analysis.
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RESULTS |
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; Thut et
al. 2003), a subset of rat trigeminal neurons (213/1282 neurons, 20.49
± 1.9%, n = 10 experiments) exhibited a substantial (i.e.,
≥20%) increase in fura fluorescence ratio in response to a decrease in
ambient temperature from 34 to 14°C. Closer inspection revealed the
existence of two major patterns of cold responsiveness
(Fig. 1, A and
B). The first group (low threshold cool,
LTcool, 98 neurons, 7.8 ± 1.1%, n = 10 experiments)
was characterized by a low threshold for activation [mean threshold = 29.0
± 1.0°C(n = 14)] and a relatively rapid increase in
intracellular calcium concentration (t1/2 = 5.6 ±
2.7 s, n = 14 neurons), whereas the second group (high-threshold
cool, HTcool, 115 neurons, 12.7 ± 2.2%, n = 10
experiments) exhibited a much higher threshold for activation (mean threshold
= 20.3 ± 0.8°C, n = 32) and a slower rise time
(t1/2 = 33.0 ± 6.5 s, n = 32 neurons,
P < 0.0005 vs. LTcool,
Fig. 1C). The
remaining 1069 neurons (79.5 ± 1.9%, n = 10 experiments)
exhibited a ≤5% change in fura ratio and were therefore designated as cold
unresponsive (UN). LTcool neurons were significantly smaller (soma
diameter 23.92 ± 0.58 µm) than HTcool neurons (26.37
± 0.90 µm, P < 0.05 vs. LTcool) or UN neurons
(26.57 ± 0.54 µm, P < 0.005 vs. LTcool,
Student's t-test). A small difference in peak response amplitudes was
measured between LTcool (55.2 ± 9.3% increase in fura ratio,
n = 14) and HTcool neurons (35.5 ± 2.5% increase,
n = 32, P < 0.05 vs. LTcool). However this
latter difference is somewhat arbitrary in nature, given that relative
intracellular calcium levels in HTcool neurons (but not
LTcool neurons) were still increasing at the time the cold stimulus
was removed (Fig. 1, B and
C). Prolonged (i.e., 5 min) stimulation of
HTcool neurons with cold typically resulted in responses that
reached a plateau within 3–4 min
(Fig. 1D).
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To gain further insight into the nature of these functionally distinct
classes of cold-responsive neurons, we examined their ability to bind the
isolectin, B4 (IB4). Small-diameter neurons can be generally divided into two
classes based on their ability to bind this isolectin. IB4-positive neurons
tend to be nonpeptidergic and project to inner lamina II of the spinal cord
dorsal horn. In the adult, the phenotype of these neurons is maintained by
glial cell line derived neurotrophic factor (GDNF). IB4-negative neurons, on
the other hand, are peptidergic and project to more superficial spinal
laminae, and their phenotype is dependent on NGF. Certain gene knockout and in
vitro electrophysiological experiments have provided evidence that these two
classes of neurons are functionally distinct
(Snider and McMahon 1998;
Stucky and Lewin 1999).
Moreover, it has been reported that in the mouse, TRPM8 mRNA is preferentially
expressed within IB4-negative neurons
(Peier et al. 2002a). In our
culture system, a large proportion of LTcool neurons failed to bind
IB4 (44/50, Fig. 2A),
whereas HTcool neurons largely did bind this lectin (37/56,
Fig. 2A,
LTcool vs. HTcool, P <
10–6). Together, these findings suggest that
LTcool and HTcool neurons differ not only in cold
responsiveness, but also in developmental subtype, in agreement with our
previous results (Thut et al. 2003).
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To further compare individual LTcool and HTcool
neurons at a molecular level, we performed single-cell RT-PCR. To validate
this technique, we first assayed the expression of mRNAs encoding molecules
known to be differentially expressed between IB4+ and IB4– sensory
neurons. mRNA encoding the ATP-gated ion channel, P2X3, which is expressed in
67–87% of IB4+ neurons
(Bradbury et al. 1998;
Zwick et al. 2002), was
detected in 16/23 IB4+ neurons, versus only 1/7 IB4– neurons (P
< 0.001). Conversely, message encoding the neurotrophin receptor, trkA,
which is expressed predominantly among IB4– sensory neurons in the adult
rat (Averill et al. 1995), was
detected in 24/58 IB4– neurons, versus only 8/48 IB4+ neurons
(P < 0.01).
We next evaluated the expression pattern of TRPM8 in the cultured neurons. TRPM8 mRNA could be detected in 23/98 cold responsive neurons (Fig. 2B). A more detailed analysis revealed that the expression of TRPM8 was significantly more prevalent among LTcool neurons (18/45) than among HTcool neurons (5/53, P < 0.0005 vs. LTcool) or UN neurons (11/87, P < 0.0005 vs. LTcool) (Table 2). Although LTcool neurons are largely IB4 negative, this feature alone could not account for the greater prevalence of TRPM8 expression observed among these cells since 8 of the 12 UN neurons in which we could detect TRPM8 were IB4 positive. The apparently low prevalence of TRPM8 expression among HTcool and UN neurons also could not be explained solely on the basis of assay sensitivity, since a virtually identical pattern was observed among 44 additional neurons in an independent experiment where input template for the PCR reaction was increased by 10-fold (Table 2).
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Because menthol can activate TRPM8 in heterologous expression systems, we
next explored the relationship between responsiveness to this agent and
expression of TRPM8 mRNA. Of 288 neurons examined, 40 (13.9%) responded to
menthol stimulation (100 µM, 60 s) with a robust, reversible increase in
relative intracellular calcium levels (data not shown). Single-cell PCR
analysis revealed that 9/33 menthol-responsive cells expressed detectable
levels of TRPM8 mRNA, whereas only 1/37 menthol unresponsive cells were TRPM8
positive (P < 0.005). Among LTcool neurons, 17/24
(70.8%) were menthol responsive, compared with 20/40 (50%) of
HTcool neurons and 2/223 (0.9%) of cold-unresponsive neurons. When
all three parameters were assayed together, TRPM8 was found in 5/15
menthol-responsive LTcool cells and in 3/16 menthol-responsive
HTcool cells. However, nearly all TRPM8-positive cells tested
(9/10) were menthol responsive, consistent with the reported pharmacological
properties of this channel protein (McKemy
et al. 2002; Peier et al.
2002a).
The correlation between TRPM8 expression and low-threshold responses to
cold stimuli, in combination with evidence suggesting that TRP channels can
heteromultimerize (Strubing et al.
2001; Tobin et al.
2002; Xu et al.
1997), led us to question whether any other TRPM family member was
associated with one or both groups of cold responsive neurons. Of the seven
other subtypes, mRNAs encoding TRPM1, TRPM3, TRPM6, and TRPM7 were detectable
at the single-cell level (Table
2). TRPM2, TRPM4, or TRPM5 mRNAs were not detected in any single
cell tested, although they were detected in samples prepared from whole TGs
(data not shown). RT-PCR revealed no association between TRPM1 or TRPM6 and
any of the three cold response patterns. TRPM3 was detected in a larger
proportion of LTcool neurons (5/28) than in all other neurons
(5/88, P < 0.05). Given the relatively low number of
TRPM3-expressing cells observed, however, we cannot exclude the possibility
that this subtype's apparently disproportionate representation represents a
sampling artifact. TRPM7 was relatively over-represented among IB4-positive
neurons (17/42 vs. 8/40 IB4 negative neurons, P < 0.05), but its
expression was not correlated with any cold response pattern
(Table 2).
Because the potassium channel TREK-1 has also been implicated as a possible
transducer of cold stimuli, we examined its expression pattern among
functionally characterized sensory neurons. TREK-1 mRNA was detected in 10/47
neurons examined. By RT-PCR, we could not detect any significant difference in
expression of TREK-1 between LTcool neurons (4/12) and
HTcool (2/13, P > 0.1 vs. LTcool) or UN
neurons (4/22, P > 0.1 vs. LTcool). This channel also
did not appear to be expressed differentially between IB4+ and IB4–
cells. Moreover, we observed no clear relationship between the expression of
TRPM8 and TREK-1 at the single-cell level. These data cannot rule out the
possibility that TREK-1 contributes to cold-transduction; however, the small
percentage of HTcool neurons expressing TREK-1 makes it unlikely
that this channel is the major cold transducer among HTcool
neurons. mRNAs encoding another candidate cold transducer (the 
,
, and 
isoforms of the ENaC channel) could not be assayed with
sufficient sensitivity to evaluate their expression among individual
cold-sensitive neurons.
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DISCUSSION |
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;
Peier et al. 2002a; Thut et
al. 2003), support a model in which TRPM8 serves as a transducer of cold
stimuli in at least a subset of LTcool neurons
(Table 3). Evidence in support
of such a model is as follows. First, both LTcool neurons and
nonneuronal cells transfected heterologously with TRPM8 respond to cooling
below 29°C with a rapid increase in intracellular calcium. Second,
LTcool neurons, similar to TRPM8-expressing neurons, are
predominantly IB4-negative. Third, we have demonstrated that TRPM8 mRNA is
detectable in a significantly larger proportion of LTcool neurons
than in HTcool or UN neurons.
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There are several possible explanations for the incomplete concordance
between TRPM8 expression and low-threshold cold responsiveness in our
experiments, as follows: 1) Alternative mechanisms for cold
transduction may exist in LTcool neurons that do not require the
expression of TRPM8. 2) TRPM8 splice variants may not be detected by
the primers chosen for our RT-PCR analysis. The existence of alternatively
spliced TRPM8 variants has not been established, although in one published
study, a TRPM8 probe recognized two distinct bands in a Northern blot of
sensory ganglion RNA (McKemy et al.
2002). It is unclear whether the additional band represents a
splice variant, or merely an incompletely spliced message. 3) Neurons
in culture may exhibit a disparity between TRPM8 mRNA and protein expression.
If so, very low levels of TRPM8 mRNA could have prevented us from detecting
the gene's expression in some cells. 4) In cold unresponsive
TRPM8-positive neurons, K+ channels or posttranslational
modifications of the TRPM8 protein might mask cold responses, as previously
suggested (Reid and Flonta
2001b; Viana et al.
2002). Immunostaining studies with TRPM8-specific antibodies may
help distinguish between these possibilities. 5) The amplification of
TRPM8 from some cells classified as cold unresponsive may indicate that these
cells expressed TRPM8 protein at a level below our calcium imaging detection
threshold had become desensitized to cold during the fura loading period, or
were otherwise functionally compromised at the time of the assay.
In our experiments, cold-responsive neurons (both LTcool and
HTcool) were more likely than the general population to respond to
menthol. However, only about one-third of menthol-responsive cells expressed
detectable TRPM8 mRNA. This difference might be explained in one of two ways.
First, it may reflect the relative sensitivity of the single-cell PCR method,
in comparison to menthol stimulation, as a predictor of TRPM8 protein
expression. Alternatively, it might indicate that menthol is capable of
activating cells not only via TRPM8, but also via one or more
TRPM8-independent mechanisms. Strong support for this latter hypothesis comes
from the fact that whereas low doses of menthol produce a sensation of
cooling, high doses of menthol exhibit irritant or anesthetic properties
(Eccles 1994). The molecule(s)
that mediates such noncooling responses might be enriched among
cold-responsive neurons, many of which appear to fall within the size range of
nociceptors.
HTcool neurons were largely TRPM8- and TREK-1-negative. These
data suggest that HTcool neurons detect cold via a
TRPM8-independent mechanism and/or that these cells express TRPM8 at a level
substantially lower than that observed in LTcool neurons. In either
case, this finding supports the idea that HTcool neurons constitute
a subpopulation of cold responsive neurons that are molecularly distinct from
LTcool neurons. As presented here and elsewhere (Thut et al. 2003),
HTcool neurons also differ from LTcool neurons in the
threshold and kinetics of their responses to cooling, cell body size, and IB4
binding. The notion that there are distinct populations of sensory neurons
responsive to a decrease in temperature is consistent with
electrophysiological and psychophysical studies, suggesting that at least two
unique populations of cold-sensitive neurons exist in vivo: one population
that is responsive to innocuous cooling and a second responsive to noxious
cold (Bessou and Perl 1969;
Darian-Smith et al. 1973;
Georgopoulos 1976;
Iggo 1969). LTcool
neurons and HTcool neurons may therefore represent cool fibers and
cold nociceptors, respectively. Moreover, differences in IB4 binding observed
in these two neuronal classes suggest that high- and low-threshold cold-evoked
responses may be differentially regulated by neurotrophins or during
pathological pain states.
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ACKNOWLEDGMENTS |
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This work was supported by grants from The W. M. Keck Foundation, Arnold and Mabel Beckman Foundation, Searle Scholars Program, and the Blaustein Pain Research Fund to M. J. Caterina, and from National Institutes of Health Grants RO3DA-13274 to M. S. Gold and T32DE-073093 to P. D. Thut.
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FOOTNOTES |
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Address for reprint requests: M. J. Caterina, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205 (E-mail: caterina{at}jhmi.edu).
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 F. Mahieu, G. Owsianik, L. Verbert, A. Janssens, H. De Smedt, B. Nilius, and T. Voets TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi J. Biol. Chem., February 2, 2007; 282(5): 3325 - 3336. [Abstract] [Full Text] [PDF]
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 K. L. Zanotto, A. W. Merrill, M. I. Carstens, and E. Carstens Neurons in Superficial Trigeminal Subnucleus Caudalis Responsive to Oral Cooling, Menthol, and Other Irritant Stimuli J Neurophysiol, February 1, 2007; 97(2): 966 - 978. [Abstract] [Full Text] [PDF]
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R. Madrid, T. Donovan-Rodriguez, V. Meseguer, M. C. Acosta, C. Belmonte, and F. Viana Contribution of TRPM8 Channels to Cold Transduction in Primary Sensory Neurons and Peripheral Nerve Terminals J. Neurosci., November 29, 2006; 26(48): 12512 - 12525. [Abstract] [Full Text] [PDF]
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 T. Taylor-Clark and B. J. Undem Transduction mechanisms in airway sensory nerves J Appl Physiol, September 1, 2006; 101(3): 950 - 959. [Abstract] [Full Text] [PDF]
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C.-K. Park, M. S. Kim, Z. Fang, H. Y. Li, S. J. Jung, S.-Y. Choi, S. J. Lee, K. Park, J. S. Kim, and S. B. Oh Functional Expression of Thermo-transient Receptor Potential Channels in Dental Primary Afferent Neurons: IMPLICATION FOR TOOTH PAIN J. Biol. Chem., June 23, 2006; 281(25): 17304 - 17311. [Abstract] [Full Text] [PDF]
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 L. Zhang and G. J Barritt TRPM8 in prostate cancer cells: a potential diagnostic and prognostic marker with a secretory function? Endocr. Relat. Cancer, March 1, 2006; 13(1): 27 - 38. [Abstract] [Full Text] [PDF]
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H. Xing, J. Ling, M. Chen, and J. G. Gu Chemical and Cold Sensitivity of Two Distinct Populations of TRPM8-Expressing Somatosensory Neurons J Neurophysiol, February 1, 2006; 95(2): 1221 - 1230. [Abstract] [Full Text] [PDF]
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E. de la Pena, A. Malkia, H. Cabedo, C. Belmonte, and F. Viana The contribution of TRPM8 channels to cold sensing in mammalian neurones J. Physiol., September 1, 2005; 567(2): 415 - 426. [Abstract] [Full Text] [PDF]
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 A. Weil, S. E. Moore, N. J. Waite, A. Randall, and M. J. Gunthorpe Conservation of Functional and Pharmacological Properties in the Distantly Related Temperature Sensors TRVP1 and TRPM8 Mol. Pharmacol., August 1, 2005; 68(2): 518 - 527. [Abstract] [Full Text] [PDF]
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B. Liu and F. Qin Functional Control of Cold- and Menthol-Sensitive TRPM8 Ion Channels by Phosphatidylinositol 4,5-Bisphosphate J. Neurosci., February 16, 2005; 25(7): 1674 - 1681. [Abstract] [Full Text] [PDF]
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D. A. Andersson, H. W. N. Chase, and S. Bevan TRPM8 Activation by Menthol, Icilin, and Cold Is Differentially Modulated by Intracellular pH J. Neurosci., June 9, 2004; 24(23): 5364 - 5369. [Abstract] [Full Text] [PDF]
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K. Tsuzuki, H. Xing, J. Ling, and J. G. Gu Menthol-Induced Ca2+ Release from Presynaptic Ca2+ Stores Potentiates Sensory Synaptic Transmission J. Neurosci., January 21, 2004; 24(3): 762 - 771. [Abstract] [Full Text] [PDF]
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