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The Journal of Neuroscience, August 1, 2002, 22(15):6408-6414
Heat-Evoked Activation of the Ion Channel, TRPV4
Ali Deniz
Güler1,
Hyosang
Lee1,
Tohko
Iida2,
Isao
Shimizu1, 3,
Makoto
Tominaga2, and
Michael
Caterina1
1 Departments of Biological Chemistry and Neuroscience,
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, 2 Department of Physiology, Mie University School of
Medicine, Tsu/Mie 514-8507, Japan, and 3 Dainippon
Pharmaceutical Company, Limited, Suita/Osaka 564-0053, Japan
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ABSTRACT |
The mammalian nervous system constantly evaluates internal and
environmental temperatures to maintain homeostasis and to avoid thermal
extremes. Several members of the transient receptor potential (TRP)
family of ion channels have been implicated as transducers of thermal
stimuli, including TRPV1 and TRPV2, which are activated by heat, and
TRPM8, which is activated by cold. Here we demonstrate that another
member of the TRP family, TRPV4, previously described as a
hypo-osmolarity-activated ion channel, also can be activated by
heat. In response to warm temperatures, TRPV4 mediates large inward
currents in Xenopus oocytes and both inward currents and calcium influx into human embryonic kidney 293 cells. In both cases these responses are observed at temperatures lower than those
required to activate TRPV1 and can be inhibited reversibly by ruthenium
red. Heat-evoked TRPV4-mediated responses are greater in hypo-osmotic
solutions and reduced in hyperosmotic solutions. Consistent with these
functional properties, we observed TRPV4 immunoreactivity in anterior
hypothalamic structures involved in temperature sensation and the
integration of thermal and osmotic information. Together, these data
implicate TRPV4 as a possible transducer of warm stimuli within the hypothalamus.
Key words:
TRPV4; OTRPC4; VR-OAC; VRL-2; TRP12; heat; ion channel; thermotransduction; osmolarity; hypothalamus
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INTRODUCTION |
The ability to detect changes in
environmental and body temperatures is critical for mammalian survival.
In the peripheral nervous system skin heating and cooling are detected
by thermosensitive neurons tuned to respond over distinct temperature
ranges. For example, warm thermoreceptors detect modest skin
temperature increases (34-42°C), whereas heat nociceptors detect
painfully hot temperatures (>42°C) (Raja et al., 1999 ). The
CNS also contains temperature-sensitive neurons, most notably in
the preoptic/anterior hypothalamus, that are activated specifically by
local warming or cooling and simultaneously receive thermally related
input from the periphery (Boulant, 2000). Although the molecular basis
of temperature sensation remains poorly understood, several ion
channels have been identified that are expressed in distinct subsets of
peripheral sensory neurons and that can be activated by changes in
ambient temperature. Among these are members of the transient receptor
potential (TRP) family, including TRPV1 (VR1) (Caterina et al., 1997)
and TRPV2 (VRL-1) (Caterina et al., 1999), which are activated by
temperatures >40 and >50°C, respectively, and the distantly related
protein TRPM8 (CMR1) (McKemy et al., 2002; Peier et al., 2002), which
is activated by cool temperatures (<25°C). These findings raise the
possibility that the transduction of thermal stimuli might be observed
among other TRP family members.
TRPV4 (OTRPC4/VR-OAC/VRL-2/TRP 12) is a nonselective cation channel
that shares ~40% amino acid identity with TRPV1 (Liedtke et al.,
2000; Strotmann et al., 2000; Wissenbach et al., 2000; Delany et al.,
2001). Expression of TRPV4 protein has been demonstrated in epithelial
cells of the renal distal convoluted tubule, trachea, and submucosal
glands, in neutrophils, and in autonomic nerve fibers (Delany et al.,
2001). In situ hybridization studies also have revealed
TRPV4 mRNA expression in hair cells of the inner ear, peripheral
sensory ganglia, and osmoregulation-related brain structures, including
the vascular organ of the lamina terminalis and the hypothalamic median
preoptic region (MnPO) (Liedtke et al., 2000; Schumacher et al., 2000).
Consistent with this localization pattern, heterologously expressed
TRPV4 can be activated by hypotonic solutions, suggesting that it
serves as a sensor for osmolarity and/or mechanical stretch (Liedtke et
al., 2000; Strotmann et al., 2000; Wissenbach et al., 2000; Delany et
al., 2001). Recently, it was demonstrated that TRPV4 also can be
activated by certain phorbol derivatives (Watanabe et al., 2002).
Although one previous study has shown that ambient temperature can
influence the magnitude of the TRPV4 response to hypo-osmolarity
(Liedtke et al., 2000), several studies have failed to detect any
activation of TRPV4 by acute changes in ambient temperature (Liedtke et
al., 2000; Strotmann et al., 2000; Delany et al., 2001). Here we
demonstrate by using two different expression systems that TRPV4 is
activated by warm temperatures and that this response is influenced by
osmolarity. Moreover, we demonstrate by using immunohistochemistry that
TRPV4 protein is expressed in the preoptic/anterior hypothalamus.
Together, these data suggest a possible role for TRPV4 in
thermosensation and/or thermoregulation.
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MATERIALS AND METHODS |
Molecular biology. An oligonucleotide
hybridization probe [5'-taagtaccccgtggtcttc-3', nucleotides (nt)
2160-2178 of the rat vanilloid receptor-related osmotically activated
channel (VR-OAC) coding region] (Liedtke et al., 2000) was used to
isolate a partial TRPV4 cDNA from a rat dorsal root ganglion cDNA
library in pCDNA3 (Invitrogen, Carlsbad, CA). Ligation of a 283 bp
5'-RACE fragment obtained from rat kidney to this partial clone
resulted in a full-length cDNA containing an open reading frame of 2612 bp identical to that of rat VR-OAC. TRPV1 cDNA in pCDNA3 has been
described previously (Caterina et al., 1999). Drosophila
TRPL (Xu et al., 1997) and human TRPC3 (Wes et al., 1995) cDNAs in
pCDNA3 were gifts of C. Montell (Johns Hopkins University, Baltimore,
MD). TRPV1 and TRPV4 cDNAs also were subcloned between the 5'- and
3'-untranslated regions of Xenopus  -globin in pXG
(gift of P. Agre, Johns Hopkins University). Unless otherwise noted,
molecular biology and cell culture reagents were obtained from
Invitrogen and chemicals from Sigma (St. Louis, MO) or Fisher
(Pittsburgh, PA).
Oocyte expression system and electrophysiology. TRPV1 and
TRPV4 cDNAs in pCDNA3 or pXG were transcribed in vitro
with T7 or T3 RNA polymerase (Epicentre Technologies, Madison, WI)
after linearization with XbaI. Stage V Xenopus
laevis (Nasco, Modesto, CA) oocytes were defolliculated with
collagenase (Worthington, Lakewood, NJ) and injected the next day with
1-50 ng of TRPV1 or TRPV4 cRNA in 50 nl of water. Oocytes were
subjected to two-electrode voltage clamp
(Eh =  40 mV) on days 2-7 after
injection via a TEV-200A amplifier (Dagan, Minneapolis, MN), PowerLab
A/D converter (AD Instruments, Mountain View, CA), and 3 M KCl-filled electrodes with a resistance of
0.4-2 M . Normal (210 mOsm) bath solution composition (in
mM) was 96 NaCl, 2 KCl, 1 MgCl2, 0.1 CaCl2, and 5 HEPES, adjusted to pH 7.4 with NaOH. Hyperosmotic (410 mOsm) bath
solution was supplemented with 200 mM mannitol.
Perfusion rate was 1 ml/min. Heat ramps from 27 to 45°C in 15 sec
were applied by preheating the perfusate with a Peltier controller
(Dagan) and were monitored with a thermistor (Physitemp, Clifton, NJ) within 2 mm of the oocyte. All procedures involving the care and use of
animals were performed in accordance with institutional guidelines.
Mammalian cell culture and calcium imaging. Human embryonic
kidney (HEK) 293/T-antigen cells maintained in DMEM/10% fetal bovine
serum/penicillin/streptomycin/L-glutamine were transiently transfected with 2 µg of plasmid DNA per 35 mm dish (including 125 ng/well green fluorescent protein (GFP) cDNA in pCDNA3) by using
Lipofectamine2000. Cells were replated onto polyornithine-coated glass
coverslips after 24 hr and subjected to calcium imaging 24 hr later.
Stable transformant TRPV1, TRPV4, and pcDNA3 cell lines were generated
by selection of transfected HEK 293 cells lacking T-antigen (gift of J. Nathans, Johns Hopkins University) with 500 µg/ml G418. Resistant
clones were screened for TRPV1 or TRPV4 surface expression by
immunofluorescence. The normal (290 mOsm) bath solution contained (in
mM) 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 1.2 NaHCO3, and 10 glucose, adjusted to pH 7.45 with
NaOH. For the experiment shown in Figure 5, this solution was adjusted
to 300 mOsm with mannitol. The 400 mOsm solution was prepared by adding
an additional 100 mM mannitol. For the 250 mOsm solution
NaCl was reduced to 105 mM. The 300 mOsm low-sodium
solution (300/M) was prepared by the addition of mannitol to the 250 mOsm solution. Osmolarities were measured by a vapor pressure osmometer
(Wescor, Logan, UT). For calcium-free solution the
CaCl2 was replaced with 10 mM EGTA.
Cells were loaded with fura-2 AM (10 µM; 37°C for 40 min) in normal bath solution containing 0.02% pleuronic acid
(Molecular Probes, Eugene, OR). Ratiometric calcium imaging was
performed with an inverted fluorescence microscope (Nikon, Melville,
NY), excitation filter changer (Sutter, Novato, CA), and cooled CCD
camera (Roper, Tucson, AZ). Paired images (340 and 380 nm excitation,
510 nm emission) were collected every 2 sec with RatioTool software
(ISee Imaging, Raleigh, NC). Transiently transfected cells were
identified on the basis of green fluorescent protein expression.
Heating was achieved as for oocyte recording and monitored with a
thermistor placed within 2 mm of the microscopic field. Target
temperatures were reached within 25-45 sec. Fura ratios were
calculated from 30 to 50 cells per coverslip, and average ratios from
multiple independent coverslips were used to calculate the sample
mean ± SEM. Unless otherwise indicated, statistical comparisons
were made with paired or unpaired Student's t tests.
Patch-clamp electrophysiology. Whole-cell patch-clamp
recordings were performed on stable transformant TRPV1, TRPV4, and
pCDNA3 cell lines as described previously (Caterina et al., 1997). The standard bath solution contained (in mM) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES,
and 10 glucose, pH 7.4 (adjusted with NaOH). The pipette solution
contained (in mM) 140 KCl, 5 EGTA, and 10 HEPES, pH 7.4 (adjusted with KOH). When examining the heat-evoked current responses,
we increased bath temperature with a preheated solution at a rate of
~0.2°C/sec. When heat-activated currents began to inactivate, the
perfusate was changed to a 22°C solution. Chamber temperature was
monitored within ± 0.1°C with a thermocouple placed within 4 mm
of the patch-clamped cell. Holding potential was 60 mV. For
current-voltage analysis, voltage-ramp pulses (100 to +100 mV) were
applied over 700 msec. Current responses obtained before heat
application were subtracted from current responses at 40°C (TRPV4,
pCDNA3) or 45°C (TRPV1).
Antibody generation and immunohistochemistry. Rabbits were
immunized with a peptide corresponding to the TRPV4 C terminus (CDGHQQGYAPKWRAEDAPL) coupled to hemocyanin (Strategic Biosolutions, Newark, DE). TRPV4 antibodies were purified from serum by peptide chromatography (Ultralink, Pierce, Rockford, IL). Antibody reactivity and specificity were confirmed by the detection of discrete protein bands in immunoblots of TPV4-transfected HEK 293 cell extracts, but not
extracts from cells transfected with pCDNA3, TRPV1, or TRPV2. Adult
male Sprague Dawley rats anesthetized with ketamine (100 mg/kg, i.p.)
and xylazine (10 mg/kg, i.p.) were perfused with PBS and then with
ice-cold 4% formaldehyde/PBS. Tissues were fixed overnight,
cryoprotected (for 48 hr in 30% sucrose/PBS), embedded in OCT (Ted
Pella, Redding, CA), and cryosectioned at 16-30 µm. Immunostaining
was performed as described previously (Caterina et al., 1999), using
affinity-purified anti-TRPV4 or anti-TRPV1 (1 µg/ml), followed by
biotinylated goat anti-rabbit IgG and nickel-enhanced diaminobenzidine
detection (Vector, Burlingame, CA) or Cy3-conjugated goat anti-rabbit
IgG (Jackson ImmunoResearch, West Grove, PA). Staining specificity was
confirmed by ablation of the signal after the incubation of antibodies
with TRPV4 peptide-conjugated resin, but not with VR1
peptide-conjugated resin.
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RESULTS |
TRPV4 mediates heat-evoked currents in
Xenopus oocytes
To evaluate TRPV4 as a possible heat transducer, we examined its
thermal sensitivity in the Xenopus oocyte expression system. Within 2-7 d the oocytes injected with complementary RNA (cRNA) encoding TRPV4 exhibited large inward currents after being heated to
45°C within 15 sec (1023 ± 103 nA at 40 mV;
n = 48) (Fig. 1B). Minimal
heat-evoked currents were observed in water-injected control oocytes
(33 ± 10 nA; n = 28; p < 0.0001) (Fig. 1A). With optimization of TRPV4
expression conditions the heat-evoked responses were observed in 87 of
90 TRPV4-injected oocytes. These currents could be produced repeatedly
in the same oocyte, with an amplitude decrement of only 17 ± 10%
over six consecutive challenges (Fig. 1C, inset).
As with TRPV4 responses evoked by hypotonic stimuli (Strotmann et al.,
2000), the inclusion of ruthenium red (100 nM) in
the perfusate during the third and fourth heat applications significantly inhibited heat-evoked currents (87 ± 2% reduction; n = 4; p < 0.001) (Fig. 1C,
inset), and these responses recovered only partially
after a 10 min washout. Neither the removal of extracellular
calcium ions nor the addition of
4,4'-diisothiocyanostilbene- 2,2'-disulfonic acid (DIDS; 500 µM) reduced the amplitude of heat-evoked TRPV4 responses (data not shown), indicating that current contamination by calcium-activated chloride flux was minimal. As described previously (Caterina et al., 1999), oocytes microinjected with cRNA encoding TRPV1
also exhibited robust inward currents at elevated temperatures, with
the initial heat-evoked response appearing at slightly higher temperatures (40-42°C) than subsequent responses (36-38°C) (Fig. 1D). By comparison, typical heat-evoked currents in
oocytes expressing TRPV4 often were detectable with temperature
elevation above ~27°C and always commenced at temperatures lower
than those required to activate TRPV1. TRPV4-expressing oocytes also
exhibited a shallower temperature-response profile than cells
expressing TRPV1. Sustained heating beyond 42°C typically resulted in
a decline in the amplitude of the TRPV4 heat-evoked current response
even as the temperature continued to rise (Fig.
1B-D). Despite this desensitization phenomenon, however, fluctuations in temperature between 36 and 42°C evoked correspondingly fluctuating current responses (Fig.
1E), as described previously for TRPV1 (Tominaga et
al., 1998). These results suggest that TRPV4 responds dynamically to
temperature changes within the physiological range.
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Figure 1.
TRPV4 mediates heat-evoked currents in
Xenopus oocytes. A-C, Two-electrode
voltage-clamp recordings. Shown are representative second
(I), fourth (II),
and sixth (III) heat-evoked current responses to
consecutive heat ramps (from 27 to 45°C in 15 sec, indicated on the
scale at the bottom) for water-injected control oocytes
(A) and oocytes injected with TRPV4 cRNA
(B, C). In C, ruthenium red
(horizontal bar, 100 nM) was added 30 sec before
the third heat application and washed away for 10 min before the fifth
heat application. Inset, Amplitudes of responses II and
III relative to response I with (open columns) or
without (filled columns) ruthenium red treatment.
Data represent the mean ± SEM; n = 4 (*p < 0.01; ***p < 0.001;
unpaired t test). D, Representative
temperature response profiles evoked by an initial heat stimulus
(top) and a second heat stimulus (bottom)
in oocytes expressing TRPV4 (V4) or TRPV1
(V1). Heat stimulus ramps went from 22 to 45°C in 15 sec. Currents were normalized to the amplitude at 45°C.
E, Effect of suprathreshold temperature fluctuations
(bottom) on representative TRPV4-mediated current
response (top).
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TRPV4 mediates heat-evoked calcium influx in HEK 293 cells
We next examined heat-evoked responses in HEK 293 cells
transiently transfected with TRPV4 cDNA under the control of a
cytomegalovirus promotor. TRPV4 expression in these mammalian cells was
evident from their elevated basal
[Ca2+]i (Fig.
2A,B) and was confirmed
by immunofluorescence microscopy (data not shown). As reported
previously (Liedtke et al., 2000; Strotmann et al., 2000),
TRPV4-expressing cells exhibited a reversible increase in
[Ca2+]i after
exposure to a hypo-osmotic medium (250 mOsm), whereas no such response
was observed in cells expressing the control vector, pCDNA3 (Fig.
2A). In response to a heat stimulus (25-40°C in
~40 sec) control cells exhibited only a modest rise in
[Ca2+]i
(0.079 ± 0.018 fura ratio units, RU; n = 5) (Fig.
2B,C). In contrast, cells transfected with TRPV4
exhibited a significantly larger rise (0.251 ± 0.033 RU;
n = 12; p < 0.001) once the
temperature exceeded ~34°C. A heat-evoked response of similar
amplitude was observed in cells transfected with TRPV1 (0.341 ± 0.08 RU; n = 3), but not in cells transfected with
either of two distantly related channel proteins, Drosophila
TRPL (0.059 ± 0.004 RU; n = 3) or human TRPC3
(0.07 ± 0.02 RU; n = 4) (Fig.
2D). This is despite the fact that both channels have
been shown to exhibit constitutive activity in mammalian cell lines (Xu
et al., 1997; Zitt et al., 1997) and that, like TRPV4-transfected
cells, TRPL-transfected cells exhibited baseline elevations in
[Ca2+]i (data not
shown).
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Figure 2.
TRPV4 mediates heat-evoked calcium influx in HEK
293 cells. A, Relative
[Ca2+]i (indicated by ratio of fura-2
emission at 340/380 nm excitation) in HEK 293 cells transiently
transfected with TRPV4 or control vector (four each) after a reduction
in osmolarity from 290 to 250 mOsm (horizontal bars) at
24°C. B, C, Relative
[Ca2+]i in HEK 293 cells transiently
(B) or stably (C)
transfected with TRPV4 or control vector (four each) during a heat
stimulus from 24 to 40°C in ~40 sec. D, Comparison
of heat-evoked (40°C) increases in fura ratio among cells transiently
transfected with control vector (P;
n = 5), TRPV4 (V4;
n = 12), TRPV1 (V1;
n = 3), Drosophila TRPL
(dT; n = 3), or human TRPC3
(hT; n = 4) or among stable
transformants generated with control vector (P;
n = 8), TRPV4 (V4;
n = 15), or TRPV1 (V1;
n = 13). Data represent the mean ± SEM
of the indicated number of coverslips. Comparisons are with controls.
*p < 0.05; ***p < 0.001;
NS, not significant; unpaired t
test.
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To rule out the possibility that the heat-evoked responses
observed in TRPV4-expressing cells were an artifact of the transient transfection method, we generated a stable HEK 293 cell line expressing this protein. Stable TRPV4 transformants had a baseline
[Ca2+]i similar to
that of control cells but exhibited an average heat-evoked [Ca2+]i increase
that was 3.4-fold as large as that observed in cells transiently
transfected with TRPV4 (Fig. 2C) and comparable with that
exhibited by stable TRPV1 transformants. Like responses to other
stimuli (Liedtke et al., 2000; Strotmann et al., 2000; Watanabe et al.,
2002), heat-evoked
[Ca2+]i increases
mediated by TRPV4 could be inhibited reversibly by ruthenium red (200 nM; 75 ± 5% reduction; n = 3; p < 0.05 vs control) or by the removal of calcium
from the extracellular medium (90 ± 3% reduction;
n = 3; p < 0.01 vs control) (Fig.
3A). In contrast, pretreatment
of TRPV4-expressing HEK 293 cells with thapsigargin (10 µM) and
1-[-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (20 µM) failed to reduce
the amplitude of the heat-evoked calcium response (data not shown).
Together, these findings suggest that the observed rise in
[Ca2+]i results
predominantly from the heat-evoked activation of calcium influx through
TRPV4 and that the release of calcium from intracellular stores does
not appear to contribute significantly to this response.
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Figure 3.
Characterization of heat-evoked calcium
responses in TRPV4-expressing HEK 293 cells A,
Reversible inhibition of heat-evoked (ramp to 40°C; horizontal
filled bars) calcium influx in cells stably expressing TRPV4 by
ruthenium red (RR; 200 nM; horizontal
open bar, top) or by removal of extracellular
calcium (horizontal open bar, bottom).
Shown at right are relative amplitudes of heat-evoked
responses during ruthenium red (hatched bars;
n = 3) or calcium-free (open bars;
n = 3) challenge (response II/response I) or after
return to normal bath solution (response III/response I). Control
(filled bars; n = 4)
represents three consecutive responses in normal bath solution.
B, Temperature response profiles of heat-evoked calcium
responses in cells stably transformed with vector (filled squares;
n = 3-4), TRPV4 (filled
circles; n = 4-11), or TRPV1 (open
circles; n = 4-12). Data represent the
mean ± SEM of the indicated number of independent microscopic
fields. C, Heat-evoked calcium responses in stable TRPV4
transformants (V4) or vector control cells
(vector; P) after acclimation (15 min) to 37°C.
Left, Representative traces from four cells of each
type. Right, Mean ± SEM increase in fura ratio
(n = 4). Data represent the mean ± SEM of the
indicated number of coverslips. Comparisons are with controls
(A, C) or between TRPV4 and TRPV1
(B). *p < 0.05;
**p < 0.01; ***p < 0.001;
unpaired t test.
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Consistent with the oocyte findings, TRPV4 expressed in HEK 293 cells
was more sensitive to heat than TRPV1 (Fig. 3B). Although TRPV4-mediated calcium responses could be observed at temperatures as
low as 34°C, TRPV1-mediated responses first became apparent only at
temperatures two to four degrees higher, with relatively modest
responses below 40°C. Two additional features of TRPV4 heat
responsiveness mirrored those observed in the oocyte system. First, the
temperature-response profile exhibited by TRPV4 was shallower than
that exhibited by TRPV1. Second, when the ambient temperature was
raised from 25 to 37°C and maintained for 15 min, the resulting
TRPV4-mediated calcium response slowly declined to baseline (data not
shown). With a subsequent increase to 42°C, however, a second calcium
response was observed that again was significantly larger than that
observed in pCDNA3-transformed control cells (Fig. 3C).
TRPV4 mediates heat-evoked currents in HEK 293 cells
Further evidence for heat-evoked activation of TRPV4 was provided
by whole-cell voltage-clamp studies of HEK 293 cells stably expressing
this protein. When cells were held at 60 mV, elevations in bath
temperature from 25 to 45°C over 80 sec resulted in inward currents
(238 ± 80 pA; n = 5) significantly larger than
any observed in pCDNA3 transformants (17.8 ± 26 pA;
n = 5; p < 0.05) (Fig. 4A). Temperature
response profiles revealed a threshold temperature for TRPV4 activation
(33.6 ± 1.8°C; n = 5) similar to that obtained in the calcium-imaging experiments (Fig. 4B).
Although the initial portion of heat-evoked TRPV4 responses exhibited
steep temperature dependence (Q10 = 9.9 ± 3.8; n = 5), the responses desensitized at
temperatures several degrees above threshold. Current-voltage analyses
performed at suprathreshold temperatures revealed a reversal potential
for TRPV4-mediated currents near 0 mV (Fig. 4C), consistent with a nonselective cationic current. Although the resulting
profiles were outwardly rectifying, as reported for
hypo-osmolarity-evoked TRPV4 responses (Liedtke et al., 2000; Strotmann
et al., 2000), the degree of rectification was substantially less than
that exhibited by heat-evoked currents mediated by TRPV1 (Rectification
ratio at ± 100 mV: TRPV4, 2.08 ± 0.44; TRPV1, 18.00 ± 6.31; n = 4).
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Figure 4.
TRPV4 mediates heat-evoked currents in HEK
293 cells. A, Representative whole-cell current traces
(at 60 mV) from HEK 293 cells stably transformed with TRPV4 or
control vector during the temperature ramp indicated at the
bottom. B, Representative
temperature-response profiles derived from cells stably transformed
with TRPV4, TRPV1, or control vector. C, Representative
current-voltage relations for heat-evoked responses in cells
transformed with TRPV4 (b), TRPV1
(c), or control vector (a).
Similar patterns were observed in four cells of each type.
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Osmolarity modulates the amplitude of heat-evoked
TRPV4 responses
Given that TRPV4 exhibits responsiveness to both heat and
hypo-osmolarity and that temperature reportedly influences
hypo-osmolarity-evoked responses in TRPV4-expressing cells (Liedtke et
al., 2000), we sought to determine whether osmolarity would impact
TRPV4 heat responsiveness. We activated TRPV4-expressing
Xenopus oocytes with two consecutive heat applications in
standard isotonic bath solution, followed by a pair of stimuli in
hypertonic bath solution (410 mOsm) (Fig.
5A). The increased osmolarity
substantially reduced the amplitude of TRPV4-mediated heat-evoked
responses (72 ± 6% reduction; n = 4;
p < 0.001). This effect was reversible, because the
response size returned to normal after reversion to an isotonic bath
solution. It was also specific to TRPV4, because TRPV1-mediated responses evoked by heat under hypertonic versus isotonic conditions were indistinguishable from one another.
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Figure 5.
TRPV4-mediated heat-evoked responses are modulated
by osmolarity. A, Representative current traces of the
second (I), fourth
(II), and sixth (III)
responses of a TRPV4-expressing oocyte subjected to consecutive heat
stimuli (45°C; horizontal open bars). Hypertonic bath
solution (410 mOsm; horizontal filled
bar) was applied 1 min before the third heat stimulus, and the
oocytes were washed for 2 min after the fourth heat stimulus.
Inset, Amplitudes of heat-evoked responses II and III
relative to response I in 410 mOsm-treated (open
columns) and untreated (filled columns)
TRPV4- and TRPV1-expressing oocytes. Data represent the mean ± SEM of four oocytes. B, Representative heat-evoked
(40°C, horizontal filled bars) calcium influx
responses in HEK 293 cells stably expressing TRPV4. Bath solution
osmolarity is indicated. The 300 mOsm low-sodium (300/M)
and 250 mOsm solutions contained equivalent NaCl concentrations and
differed only by the presence or absence, respectively, of mannitol. In
the bottom two traces the experiment was initiated in
300/M solution and was switched to 250 mOsm at the time
indicated by the arrows. C, Summary of
heat-evoked calcium responses at indicated osmolarities for cells
stably transformed with control vector (P; open
columns; n = 3-4) or TRPV4
(V4; filled columns;
n = 5-9). Data represent the mean ± SEM of
the indicated number of coverslips. Comparisons in A and
C are with the appropriate 300 mOsm or 300/M
controls. *p < 0.05; **p < 0.01; ***p < 0.001 (NS, not
significant; unpaired t test).
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A similar dependence of heat-evoked activation of TRPV4 on osmolarity
was observed in the HEK 293 expression system. Under hyperosmotic
conditions (400 mOsm) stable TRPV4 transformants exhibited a reduction
in their mean calcium influx response to heat (40°C) compared with
control TRPV4 cells that were heat stimulated at 300 mOsm (0.10 ± 0.01 RU, n = 6 vs 0.47 ± 0.03 RU,
n = 5; p < 0.0001) (Fig.
5B,C). After restimulation with heat under isotonic conditions, the cells initially heated in the hypertonic solution exhibited larger heat-evoked responses resembling those of isotonic controls (data not shown). A decrease in osmolarity to 250 mOsm (at a
constant extracellular sodium concentration) produced a hypo-osmolarity-evoked response that reached a peak within ~2 min. A
heat stimulus applied at this point evoked a superimposed calcium
response that was significantly larger than that observed at 300 mOsm
(1.54 ± 0.33 RU, n = 6 vs 0.59 ± 0.07 RU,
n = 6; p < 0.05). Indeed, even if the
hypo-osmolarity-evoked response was allowed to desensitize to baseline
before heat challenge (after 5-15 min), a large heat-evoked response
was still observed (1.23 ± 0.25 RU; n = 9;
p < 0.05 vs 300 mOsm response). Together, these data
argue strongly that heat and osmolarity can act in concert on TRPV4 to
regulate its activity, but that even after the hypo-osmolarity-evoked response of TRPV4 has undergone desensitization, substantial
responsiveness to heat persists.
TRPV4 is expressed in thermosensory regions of
the hypothalamus
In an attempt to identify anatomical structures in which the
coordinate detection of osmolarity and temperature by TRPV4 might be
physiologically relevant, we performed TRPV4 immunolocalization in rat
tissues. Among a number of brain regions noted to exhibit specific
TRPV4 immunoreactivity was the preoptic/anterior hypothalamus, most
notably the medial preoptic area (MPA) (Fig.
6A) and MnPO (Fig.
6B). Although we detected TRPV4 mRNA in dorsal root
and trigeminal ganglia by Northern blot and RT-PCR, we
observed no specific TRPV4 immunoreactivity in the
cell bodies of these ganglia (data not shown). We did, however, observe
intense TRPV4 immunoreactivity within suprabasal keratinocytes of
plantar skin (Fig. 6C,D).
View larger version (93K):
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|
Figure 6.
Immunohistological detection of TRPV4 in the
anterior hypothalamus and skin keratinocytes. A-D,
TRPV4-specific diaminobenzidine immunostaining of the MPA (A,
B) and MnPO (C, D) regions of rat hypothalamus.
In B and D, anti-TRPV4 was predepleted
with antigenic TRPV4 peptide. AC, Anterior commissure;
3V, third ventricle. E, F, TRPV4
immunofluorescence in rat plantar skin keratinocytes
(arrow) and sweat glands (arrowhead). In
F, anti-TRPV4 was predepleted with the antigenic TRPV4
peptide.
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|
| |
DISCUSSION |
We have found that heterologously expressed TRPV4 mediates
heat-activated current influx in Xenopus oocytes and HEK 293 cells. In oocytes TRPV4 activation becomes apparent at temperatures
>27°C, whereas in the HEK 293 cells the threshold for activation is
~34°C. In both systems, therefore, TRPV4 can be activated by
modestly elevated (i.e., warm) temperatures lower than those required
to activate TRPV1. Prolonged suprathreshold heat stimuli cause the TRPV4-mediated response to desensitize, a phenomenon reported previously for TRPV1 (Caterina et al., 1999). Of potential
physiological importance, however, is our observation that, once
TRPV4-expressing HEK 293 cells have been acclimated at 37°C, TRPV4
remains capable of mediating responses to further increases in ambient temperature.
It is unclear why several other investigators have failed to
observe the heat-evoked activation of TRPV4 (Liedtke et al., 2000;
Strotmann et al., 2000; Delany et al., 2001). One possible explanation
stems from our observation that, in HEK 293 cells transiently
expressing TRPV4, a substantial fraction of TRPV4 immunoreactivity
resides within an apparently intracellular compartment, whereas in
stable TRPV4 transformants nearly all TRPV4 apparently is associated
with the plasma membrane (data not shown). The incomplete surface
expression of TRPV4 in transiently transfected cells is associated with
smaller mean heat-evoked calcium responses than those observed in
stable transformants. Moreover, heat stimulation produces a
"background" calcium increase in control vector transfected cells
that becomes relatively large above 45°C. Because hypo-osmolarity produces no such background, small heat-evoked responses might have
been masked out of proportion to hypo-osmolarity-evoked responses in
those studies in which heat-evoked responses were evaluated in
transiently transfected cells (Strotmann et al., 2000; Delany et al.,
2001). Other factors that may have impaired the detection of
heat-evoked responses include the use of GFP-tagged constructs (Strotmann et al., 2000) or the desensitization of heat-evoked responses during slow temperature ramps with infrequent sampling (Liedtke et al., 2000). Finally, cofactors necessary for heat-evoked activation of TRPV4 might be expressed differentially among host cells.
Single-channel analyses of TRPV1 in excised membrane patches have
suggested a direct effect of heat on the open probability of that
channel (Tominaga et al., 1998). In the absence of similar analyses of
TRPV4, we cannot yet rule out the possibility that this protein is
activated indirectly by other heat-stimulated proteins, by small
molecules liberated in response to heat, or by the physicochemical
effects of heat on the plasma membrane. Indeed, an indirect mechanism
previously has been suggested for certain heat-evoked responses in
cultured sensory neurons (Reichling and Levine, 1997).
Our findings also support the functional integration of thermal and
osmotic information at TRPV4. Heat-evoked TRPV4 responses are larger at
relatively low osmolarities and virtually ablated at high osmolarities.
Previous studies have suggested the converse relationship, namely that
the hypo-osmolarity response of TRPV4 is temperature-dependent, with a
greater response being observed at 37 than at 22°C (Liedtke et al.,
2000). These two findings are likely to reflect the same stimulus
convergence process. However, our observation that heat-evoked
responses of full size can be evoked after desensitization of a
hypo-osmolarity-evoked response suggests that some degree of
independence exists between these two mechanisms of TRPV4 activation.
An analogous situation has been observed for TRPV1, in which proton-,
heat-, and capsaicin-evoked responses are mechanistically
distinguishable, although they interact (Jordt et al., 2000; Jordt and
Julius, 2002). As with heat, the mechanism by which hypo-osmolarity
activates TRPV4 is unknown. In particular, it is not entirely clear
whether TRPV4 senses hypo-osmolarity, per se, the resultant cellular
swelling, or a signaling molecule released in response to swelling.
Regardless, these findings and the recently demonstrated activation of
TRPV4 by phorbol derivatives (Watanabe et al., 2002) suggest that this
protein, like TRPV1, can be regulated by an array of partially
convergent physical and chemical stimuli.
Under what physiological circumstances might the thermal activation of
TRPV4 be relevant? The expression of TRPV4 protein in the MPA and MnPO
regions of the anterior hypothalamus, in addition to its
temperature-response characteristics, suggests that it might act as a
heat transducer in this setting. Electrophysiological studies of
organotypic anterior hypothalamic slices have provided evidence for the
existence of a heat-activated nonselective cation channel in
warm-sensitive neurons, although the molecular identity of that channel
is not known (Hori et al., 1999). Such studies also have revealed a
complex relationship between thermosensation and osmosensation at the
level of individual anterior hypothalamic neurons. Although many
warm-sensitive neurons are inhibited by local hyperosmolarity or
activated by hypo-osmolarity, others exhibit the opposite relationship
(Silva and Boulant, 1984; Travis and Johnson, 1993). Given its dual
responsiveness to thermal and osmotic stimuli, TRPV4 might mediate some
of these composite effects. Consistent with reported results (Liedtke
et al., 2000; Schumacher et al., 2000; Delany et al., 2001), we
detected TRPV4 mRNA in peripheral sensory ganglia but failed to observe
TRPV4 immunoreactivity in the neuronal cell bodies of these ganglia.
Therefore, it is unclear to what extent this protein is expressed
in primary afferent neurons or whether it contributes to peripheral
heat transduction. We did observe specific TRPV4 immunoreactivity in
skin keratinocytes. No direct role in thermosensation has been
demonstrated for these cells. Nevertheless, their proximity to the
animal's surroundings and to the nerves that transmit thermal and
mechanical information to the CNS makes this observation intriguing,
especially in light of the report that TRPV1 also is expressed in
keratinocytes (Inoue et al., 2002). Given these findings, future
studies should be aimed at evaluating the functional significance of
TRPV4 heat sensitivity, osmosensitivity, and expression in
neuronal and non-neuronal cells by using native preparations. In
addition, the polymodal responsiveness of TRPV4 creates an opportunity
to use structure-function approaches to explore the biophysical basis
of thermosensation and osmosensation. Such studies may reveal why
responsiveness to one or both of these modalities is an increasingly
common feature of the TRPV subfamily.
| |
FOOTNOTES |
Received Feb. 15, 2002; revised May 20, 2002; accepted May 20, 2002.
This work was supported by American Cancer Society Grant
RGS-01-063-01-CSM; by awards from the W. M. Keck Foundation,
Searle Scholars Program, and Arnold and Mabel Beckman Foundation and a
gift from Dainippon Pharmaceuticals to M.J.C.; and by grants from the
Ministry of Education, Culture, Sports and Technology, Japan to M.T. We
thank J. Wang and R. Evans for technical assistance and P. Coulombe, D. Johns, M. Nealen, and C. Montell for valuable suggestions.
Correspondence should be addressed to M. J. Caterina, Departments
of Biological Chemistry and Neuroscience, 725 North Wolfe Street, Johns
Hopkins University School of Medicine, Baltimore, MD 21205. E-mail:
caterina{at}jhmi.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
