From the Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77030
Received for publication, March 12, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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A recent member of the TRPV subfamily, TRPV4 (also called OTRPC4, TRP12,
and VR-OAC), has been cloned and expressed in a variety of heterologous
expression systems
(1518).
Initial evidence indicated that the channel may function as a mammalian
osmoreceptor and hence may play a central role in osmoregulatory functions
(1618).
However, the Caenorhabditis elegans homologue, OSM-9, has been shown
to underlie a wide range of sensory functions, including olfactory,
mechanosensory, and osmosensory processes
(19,
20). Further, in mouse, rat,
human, and chicken, TRPV4 has been shown to be distributed in a broad range of
tissues, including trachea, kidney, liver, heart, lung, testis, brain, and
ear, implicating a potentially diverse range of sensitivities and functions
for the mammalian channel. Although initial studies demonstrated that this
channel may only be activated by hypotonicity
(1618)
and not by mechanical stresses such as membrane stretch
(17), more recent studies have
demonstrated that it can also be activated by phorbol ester derivatives, such
as 4-PDD,1 that
do not activate protein kinase C
(21). Early studies indicated
that the mammalian TRPV4 channels appeared to have a much narrower range of
sensitivities to mechanical and, possibly, chemical stimuli than their
counterparts in C. elegans or the first member of the subfamily,
TRPV1. However, subsequent studies have recently demonstrated that the
mammalian TRPV4 channels may be sensitive to temperature, displaying a
temperature-dependent activation between 30 and 43 °C
(22,
23). Because temperature is a
dominant modulator of biological processes, it may be that temperature also
serves to modulate the gating properties of the mammalian TRPV4 homologues to
enhance its sensitivity to activation by microenvironmental
physical/mechanical and chemical stimuli.
The purpose of the present study was to determine whether temperature plays a critical role in modulating the sensitivity of the TRPV4 channel to microenvironmental chemical and physical stresses or stimuli. The TRPV4 channel was cloned from mouse M-1 kidney cells and expressed in heterologous expression systems. Elevation of the temperature from 2224 to 37 °C was found to impart a critical sensitivity to this channel for chemical and physical stimuli, uncovering a diversity of gating signals. Indeed, at 37 °C the channel was found to be highly sensitive to osmotic stress, as shown by others, and to mechanical stress (shear stress). In addition, the channel was shown to be sensitive to phorbol ester derivatives but through two divergent transduction pathways, namely a PKC-dependent pathway and a PKC-independent pathway. Pharmacological characterization of the channel displayed potent blockage by ruthenium red but not by Gd3+ or La3+. Parts of this study have been presented in abstract form (24).
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TRPV4 ExpressionHEK 293 cells and Chinese hamster ovary (CHO) cells were used. HEK 293 cells were grown in standard Dulbecco's modified Eagle's medium/Ham's F-12 medium at 37 °C (with 2 mM L-glutamine), pH 7.4, and CHO cells were grown in modified Ham's F-12 medium at 37 °C, pH 7.4, containing 10% fetal bovine serum (Sigma).
The pcDNA3.1/TRPV4-V5 plasmid was initially transiently transfected into
HEK 293 cells and CHO cells using the lipid-mediated LipofectAMINETM 2000
kit (Invitrogen). Transfections using both positive controls, pcDNA3.1/V5-His
vector containing a LacZ cDNA insert with the fused V5 epitope, and
negative controls, without an insert, were performed to verify the cloning and
transfection procedures. With transient transfections for 4872 h,
3060% of the HEK 293 cells and CHO cells were transfected based on
immunohistochemical identification of the number of cells expressing the V5
epitope (see "Immunohistochemistry and Immunoblotting" below). To
facilitate the study, stably transfected cells were established by selection
of transfected cells with Geneticin (Invitrogen). The stable transfected cell
lines were evaluated using immunohistochemical identification based on the
expression of the V5 epitope and functional identification of cells positively
responding (intracellular calcium change) to channel stimuli (e.g.
hypotonic medium and 4
-PDD) as established by others (Refs.
16 and
21; also see below).
Functional evaluation of TRPV4 activation, using known channel stimuli to
activate the channel (e.g. hypotonic medium or 4
-PDD),
likewise demonstrated cells expressing the channel.
Immunohistochemistry and ImmunoblottingImmunohistochemical localization of expressed TRPV4 protein with the C-terminal V5 epitope was assessed using an anti-V5 antibody conjugated to FITC (anti-V5-FITC; Invitrogen). Briefly, the cells were grown on coverslips, washed with PBS, fixed in 100% methanol (room temperature) for 5 min, blocked with 1% bovine serum albumin/PBS, and then incubated with anti-V5-FITC antibody diluted (1:500) in 1% bovine serum albumin/PBS for 1 h. After washing with PBS, VectraShield mounting medium (Vector Laboratories) was applied, the coverslip was placed on a glass slide, and the cells were visualized on a Nikon Diaphot epifluorescence microscope (FITC filter set) and digitally photographed.
To provide further assessment of TRPV4 expression, immunoblotting of cell homogenates was performed as previously described (25), taking advantage of the C-terminal V5 epitope. Total protein aliquots (510 µg) were separated by 6% SDS-PAGE and transferred onto activated polyvinylidene difluoride membranes (Amersham Biosciences). After transfer, the polyvinylidene difluoride membranes were blocked with Tris-buffered saline/Tween 20 (0.05%) containing 5% nonfat milk for 1 h with gentle rocking. The anti-V5 antibody conjugated to horseradish peroxidase (anti-V5-HRP; Invitrogen) was applied (1:1000) in blocking buffer for 1 h, then washed with Tris-buffered saline/Tween 20 (0.05%), and visualized on ECL Hyperfilm using an ECL detection reagent chemiluminescence system (Amersham Biosciences).
Measurement of Intracellular CalciumThe intracellular
calcium levels ([Ca2+]i) were quantitatively
monitored in cultured cells using the fura-2 fluorescence technique as
described previously (26,
27). Briefly, HEK and CHO
cells grown on coverslips were loaded with fura-2 by incubation of the cells
with 510 µM fura-2 acetoxymethyl ester in culture medium
for 1 h at 37 °C. The cells were washed with isotonic medium (ISO, see
"Solutions and Chemicals" below) and incubated an additional
1015 min to abolish compartmentalization of fura-2/AM in cells. The
coverslips, with fura-2-loaded cells, were attached to the bottom of a custom
designed perfusion chamber (1.5 ml) or a shear stress chamber (see below) as
appropriate, and the chamber was attached to the microscope stage of our InCa
Imaging Work station (Intracellular Imaging, Inc.). The temperatures were
maintained by continuous flow through of heated bathing solutions or by
exchange of preheated bathing solutions. The cells were bathed in either ISO
or hypotonic (HYPO) medium as defined below (see "Solutions and
Chemicals"). Typically 1030 cells were simultaneously monitored
on a coverslip, and the results were averaged for each experiment.
Intracellular calcium was estimated from the fura-2 fluorescence by excitation
at 340 and 380 nm and calculating the ratio of the emission intensities at 511
nm in the usual manner every 13 s. The fura-2 fluorescence ratios were
converted to intracellular calcium activity
([Ca2+]i) as described by Grynkiewicz et
al. (28).
![]() | (Eq. 1) |
Shear StressFor measurement of the effects of shear stress on TRPV4 channel activation, the cells grown on coverslips were attached to the bottom of a Flexcell Flow chamber (Flexcell International, Inc.), and the chamber was attached to the microscope stage of the InCa Imaging Work station as described above. Solution (ISO or HYPO) flow through the chamber was regulated by a peristaltic pump (075 ml/min) to generate shear stresses from 0 to 20 dyne/cm2 while simultaneously monitoring intracellular calcium levels in individual cells (typically 1030 cells), and the results were averaged for each coverslip.
Patch Clamp Current MeasurementsThe patch clamp technique
in whole cell configuration was used for measurements of whole cell currents
using an Axon Instruments Axopatch-1D voltage clamp (with Bessel filter, 2
kHz) interfaced to a Digidata 1322A data acquisition system using pClamp 8
software (Axon Instruments) as done previously
(36). Patch pipette
electrodes, fabricated with a Narashige PP-83 micropipette puller and MF-83
Microforge, had resistances of 24M (see pipette solution below
under "Solutions and Chemicals"). The reference electrode was a 3
M KCl agar bridge inserted into a continuously flowing bath
solution downstream from the patch electrode. Series resistance was
compensated electronically by 4080%. The temperature of the
electrophysiological bathing solution (see "Solutions and
Chemicals" below) was maintained at 37 °C using a direct
current-regulated in-line heating jacket (Warner Instruments) to warm the
bathing solution as it entered the study chamber. Typically all current
recording was done at a holding potential of -60 mV (sampling rate of 5 kHz,
filtered at 2 kHz) with a voltage ramp protocol applied every 10 s as done by
others (21). The voltage ramp
protocol was initiated by a voltage step to -100 mV for 20 ms, followed by a
400-ms ramp from -100 to +100 mV (sampling interval of 0.2 ms) and returning
to the holding potential of -60 mV.
Solutions and ChemicalsHEK 293 or CHO cells were washed and bathed in modified balanced salt solutions for all functional studies assessing intracellular calcium levels. Cells were either bathed in an ISO or HYPO as appropriate. The ISO contained 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 0.4 mM MgSO4, 3.3 mM NaHCO3, 2.0 mM CaCl2, 10 mM HEPES, 5.5 mM glucose, pH 7.4, having an osmolarity of 300 mOsm/liter. The HYPO was identical, except the NaCl concentration was reduced to 100 mM, reducing the osmolarity to 225 mOsm/liter. For electrophysiogical studies measuring whole cell currents, the extracellular bathing medium was 150 mM NaCl, 1 mM MgCl2, 5 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4. The patch pipette solution was 100 mM cesium aspartate, 20 mM CsCl, 1 mM MgCl2, 4 mM Na2ATP, 0.037 mM CaCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.2.
PMA (Sigma), a PKC-activating phorbol ester, and 4 -PDD (Sigma), a
non-PKC-activating phorbol ester, were used at 100 nM (100
µM stock in Me2SO), and RR (Sigma), a channel
blocker, was used at concentrations of 110 µM (10
mM stock in water). The drugs were diluted in medium and sonicated
just before use. Cal C (Calbiochem), a relatively selective inhibitor of PKCs,
and staurosporine (Sigma), a broad spectrum PKC inhibitor, were used at
concentrations of 50 nM (0.1 mM stock in
Me2SO) and either 50/500 nM (1 mM stock in
Me2SO), respectively. All other chemicals were from Sigma unless
otherwise noted.
StatisticsIntracellular calcium levels were measured in
individual cells for each experiment on a given coverslip, and the results
were averaged to provide a single experimental run. Hence, for this study
n is the number of experimental runs (i.e. number of
coverslips). For electrophysiological studies, each current measurement was
from a single cell. The pooled data are given as the means ± S.E. The
significance differences between groups were tested with Student's paired or
unpaired t test, as appropriate. p 0.05 was assumed to
be significant.
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RESULTS |
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The TRPV4 protein was also characterized by immunoblotting, again taking advantage of the V5 epitope construct and the anti-V5-HRP antibody for detection. As shown in Fig. 1 (C and F), a single protein band was detected in both transfected HEK 293 and CHO cells, whereas no bands were evident in nontransfected cells. The molecular mass of the band in both transfected HEK 293 and CHO cells was estimated to be near 100 kDa. Because this construct includes the fused C-terminal segment containing the V5 epitope and accessory domains, the actual TRPV4 protein size on immunoblots is near 95 kDa.
Effect of Temperature on Basal Calcium LevelsThe functional properties of the TRPV4 channel were assessed from measured changes in intracellular calcium levels, [Ca2+]i, following channel activation. Measurement of [Ca2+]i levels in these cells using the fura-2 fluorescent methods was uncomplicated in the present study because expression of the TRPV4 construct with the V5 epitope does not produce a fluorescence tag and hence does not interfere with the fura-2 (or fluo-3) signal, in contrast to that observed for the green fluorescent protein tag (29). Initial studies focused on the effect of changing the study temperature from room temperature (2224 °C) to 37 °C without the addition of known channel activators. As shown in Fig. 2, at room temperature [Ca2+]i levels in nontransfected and TRPV4-transfected cells were low, averaging 35 ± 4 and 44 ± 4 nM for nontransfected and transfected HEK 293 cells, respectively, and 48 ± 14 and 58 ± 7 nM for nontransfected and transfected CHO cells, respectively. At 37 °C, [Ca2+]i levels were elevated for both nontransfected and transfected cells, averaging 82 ± 10 and 86 ± 12 nM for nontransfected and transfected HEK 293 cells, respectively, and 103 ± 6 and 108 ± 14 nM for nontransfected and transfected CHO cells, respectively. Hence, under the conditions of the present studies where cells were incubated at two different stable temperatures, transfection alone did not appear to result in a measurable change in the basal, steady-state [Ca2+]i levels, although considerable cell-to-cell variability was evident. In all subsequent experiments, the effect of temperature on TRPV4 activation was evaluated primarily in HEK 293 cells, with only a few confirmatory studies performed in CHO cells.
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Temperature-modulated Gating of TRPV4 by Phorbol EstersIt
has been shown previously that phorbol ester derivatives, such as PMA and
4-PDD, can activate TRPV4 by a PKC-independent mechanism
(21). Because these earlier
studies were done at room temperature, an effect of elevated temperature on
the lipid signals may modulate TRPV4 gating sensitivity. PMA, in particular,
is a relatively specific activator of PKC at low concentrations of 100
nM or less, whereas the actions of 4
-PDD are
PKC-independent. Hence, the effects of temperature on the actions of both PMA
and 4
-PDD at 100 nM were assessed.
In studies at room temperature in transfected HEK 293 cells, the
application of 4-PPD (100 nM) produced a modest transient
increase in [Ca2+]i in each study to a peak
value of 166 ± 91 nM
(Fig. 3, A and
B). At 37 °C, the addition of 4
-PDD produced a
more robust response with [Ca2+]i increasing to
a peak value of 622 ± 93 nM. At elevated temperatures the
[Ca2+]i response appeared to reflect a simple
enhancement of the response observed at room temperature. No significant
response of 4
-PDD was observed in nontransfected HEK 293 cells (data
not shown).
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The [Ca2+]i response to PMA (100 nM) was highly dependent upon temperature. At room temperature, the addition of PMA in transfected HEK 293 cells produced a minimal response, with [Ca2+]i increasing to only 62 ± 14 nM (Fig. 3, C and D). In contrast, at 37 °C PMA addition now produced a potent response with [Ca2+]i increasing to a peak value of 483 ± 98 nM. Hence, the effect of PMA went from essentially no response at room temperature to a very potent response at elevated temperatures.
Temperature-modulated Gating of TRPV4 by Physical StressesPhysical stress in the form of hypotonic swelling is a known activator of the TRPV4 channel. It was observed in the present study that when cells were subjected to hypotonic swelling at room temperature, only a modest increase in [Ca2+]i was observed in transfected HEK 293 cells, increasing from near 44 ± 6 to 130 ± 30 nM (Fig. 3, E and F). When cells were studied at 37 °C, however, hypotonic swelling now produced a pronounced change, with [Ca2+]i increasing from 74 ± 13 to 406 ± 74 nM. These changes were not observed in nontransfected cells (data not shown).
To assess further the potential role of physical stress on TRPV4 gating, the studies were expanded to evaluate the effects of fluid shear stress, attributable to changes in fluid flow rate, on channel activation. At room temperature in transfected HEK 293 cells, increasing the fluid shear stress from 0 to 10 dyne/cm2 had little or no effect on [Ca2+]i (Fig. 3, G and H), a finding consistent with a lack of response to membrane stretch at room temperature as shown by others (17). In contrast, when studies were done at 37 °C, increasing fluid shear stress to 10 dyne/cm2 resulted in a marked increase in [Ca2+]i to a peak value of 185 ± 25 nM, often resulting in [Ca2+]i oscillations (see example in Fig. 3G). At 37 °C the channel response was tightly regulated by the magnitude of shear stress applied. Step elevations in shear stress from 0 to 3, 10, or 20 dyne/cm2 resulted in proportional increases in [Ca2+]i (Fig. 3H, inset) with a strong positive correlation (r = 0.920, p < 0.01). Hence, these studies demonstrate that elevated temperatures are required to link TRPV4 channel activation to shear stress, results again consistent with a temperature-induced potentiation or switching on of a channel signaling pathway.
PKC-dependent and -independent Pathways Regulate TRPV4
GatingSome TRP channels are activated by release of calcium from
internal stores. This does not appear to be the case for TRPV4 activation by
physical and chemical stimuli. At 37 °C, removal of calcium from the
extracellular medium completely abolished any
[Ca2+]i rise upon the addition of 4-PDD
or PMA (Fig. 4,
AD). Likewise, removal of extracellular calcium
abolished any rise in [Ca2+]i following
exposure to HYPO (Fig. 4, E and
F). None of the stimuli would appear to induce the
release of calcium from the endoplasmic reticulum calcium stores. Hence, the
transduction pathways controlling gating of TRPV4 do not appear to involve
store-operated signals or release of calcium from storage sites.
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To assess potential transduction pathways that may regulate TRPV4
activation, a potential role of PKC was evaluated at 37 °C. First, the
addition of 4-PDD, a non-PKC-activating phorbol ester, induced a potent
[Ca2+]i response in each case, producing a peak
change in [Ca2+]i of 1108 nM
(Fig. 4, A and
B) similar to that observed earlier. If cells were first
treated with Cal C (50 nM), a selective inhibitor of PKC, the
subsequent addition of 4
-PDD was not inhibited with the peak change in
[Ca2+]i of 803 nM
(Fig. 4, A and
B, p > 0.6, paired). This is as anticipated
because 4
-PDD does not activate PKC.
Because PMA is a known activator of PKC, particularly at the modest levels of 100 nM used in the present study, its effects may be acting through a PKC-dependent pathway. Again, the addition of PMA produce a rapid response in [Ca2+]i with a peak change in [Ca2+]i of 368 ± 51 nM (Fig. 4, C and D). If PKC were first inhibited by addition of Cal C (50 nM), the actions of PMA addition on [Ca2+]i were dramatically reduced, with the peak change averaging only 48 ± 18 nM (Fig. 4, C and D, p < 0.001, paired). Likewise, if PKC were inhibited by the addition of staurosporine (50 or 500 nM), a broad spectrum PKC inhibitor, instead of Cal C, the effect of PMA on [Ca2+]i was markedly reduced with peak changes averaging 91 ± 22 nM (n = 4) and 49 ± 32 nM (n = 4) with 50 and 500 nM staurosporine, respectively. Hence, the actions of PMA are largely abolished by PKC inhibition.
The mechanism of action of hypotonic swelling on TRPV4 activation is not known. To determine a potential role for PKC in this process, the effects of inhibition of PKC by Cal C treatment (50 nM) on the [Ca2+]i response to HYPO were evaluated. As shown in Fig. 4 (E and F), Cal C treatment had little or no effect on the HYPO induced [Ca2+]i changes. The actions of HYPO treatment on TRPV4 gating would appear to be primarily through transduction pathways not directly linked to PKC.
Pharmacological Characterization of TRPV4To further
characterize the channels activated by the physical and chemical stimulants in
the present study, potential pharmacological channel blockers were evaluated
for each of the stimulants. Traditional blockers of calcium-permeable
channels, such as La3+ and Gd3+ (up to 500
µM), were not effective blockers, often leading to an initial
apparent calcium influx as reported by others for TRPV4
(16). Recently, RR has been
shown to block TRPV4 channels
(17,
21,
22). RR was found to be an
effective blocker of TRPV4 in the present study (110 µM)
and provided a tool to functionally characterize the channels activated by
each of the stimuli. The inhibitory actions of RR were relatively potent, with
1 µM RR effectively blocking the channel. As shown by the
example in Fig. 5A,
the addition of RR prior to activation of the channel essentially abolished
channel gating by 4-PDD. Similar results were found for RR actions with
PMA, HYPO, and shear stress (examples not shown). If RR were added after
channel activation, its actions were relatively rapid, although the effect did
not appear to be as potent. Nonetheless, RR appeared to generate a similar
block of calcium entry for all physical and chemical stimuli. Indeed, the
addition of RR (1 µM) was found to potently reduce the rise in
[Ca2+]i for 4
-PDD, PMA, HYPO, and shear
stress by 96, 92, 88, and 68%, respectively
(Fig. 5, BE).
Hence, RR had a similar blocking action for all physical and chemical
stimulants as anticipated if all stimulants activated the same TRPV4
channel.
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Patch Clamp Analysis of TRPV4 CurrentsTo verify that the
actions of PMA on intracellular calcium levels at 37 °C were indeed due to
activation of current flow through TRPV4 channels, as previously shown for
HYPO and 4-PPD by others
(12,
16,
21), the effects of PMA on
whole cell currents in TRPV4-transfected and nontransfected cells was assessed
and compared with that obtained with 4
-PDD at 37 °C. In
nontransfected cells, neither 4
-PPD nor PMA produced an increase in
whole currents as shown in Fig. 6
(A and B), respectively. In contrast, both
4
-PPD and PMA induce significant inward currents (cation influx) in
TRPV4-transfected cells (Fig. 6, C
and D), averaging near -100 pA for both phorbol esters
(Fig. 6G). The
current-voltage relations displayed the typically outward rectification
characteristic of the TRPV4 channel
(17,
21) with outward currents
(positive currents) increasing strongly with positive voltages
(Fig. 6, E and
F). The addition of ruthenium red (1 µM)
abolished the 4
-PPD- and PMA-activated currents (data not shown) as
expected, whereas the addition of 100 nM calphostin C to the
bathing medium 5 min prior to addition of a phorbol ester inhibited the
PMA-induced current (75% inhibition, n = 5) but not the
4
-PPD-induced current (data not shown). Hence, the electrophysiological
recordings confirm that PMA does indeed lead to activation of the TRPV4
channel when studied at 37 °C.
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DISCUSSION |
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A Molecular Integrator of Microenvironmental SignalsThe mechanism of activation of the TRPV4 channel remains unknown. With the present demonstration that elevated temperatures may link the gating of the channel to a diverse range of signals, it is likely that multiple mechanisms or signaling pathways may underlie regulation of the channel giving rise to the diversity of gating signals. Because hypotonic swelling and shear stress both potently regulate channel gating, the channel likely has both osmosensory and mechanosensory functions. The mechanism(s) by which physical signals regulate the channel, however, remains unknown. Nonetheless, having several physical signals converging on the same channel likely indicates that the channel is a site of signal integration.
The separate identification that phorbol ester derivatives may activate the channel through both PKC-dependent and -independent transduction pathways (Fig. 4, A and B) raises the possibility that the channel may also be regulated by multiple endogenous ligands. Many ligands, such as peptide hormones, often signal through the ubiquitous phospholipase C-PKC transduction pathways that generate diacylglycerol, activate PKCs, and, in turn, activate downstream effectors (30). The concept of a parallel transduction pathway involving phorbol esters/diacylglycerol in a PKC-independent manner is less well known, but such pathways are increasingly being discovered. Indeed, it is now known that numerous signaling proteins have C1 domains that bind phorbol esters/diacylglycerol in PKC-independent manner and play critical roles in regulating signaling functions (31). Evidence of such mechanisms regulating calcium and potassium ion channels has also been forthcoming in recent years (3234). It seems highly likely, therefore, that although endogenous ligands activating the channel have not yet been identified, they are soon to be discovered. The results of the present study should aid in these investigations. Regardless of the mechanisms, however, the gating diversity of the TRPV4 channel demonstrated in the current study points to the presence of multiple physical and chemical signaling pathways that converge on the channel. As a result, the TRPV4 channel would appear to function as a molecular integrator of a complex array of diverse signals.
The diversity of channel regulatory behavior observed for the TRPV4 channel appears to have components similar to that observed for the founding member of the vanilloid channels, the vanilloid receptor TRPV1. TRPV1 is activated by a wide range of physical and chemical signals including noxious heat, acidity, anandamide, and capsaicin, demonstrating a remarkable diversity in gating signals and, like TRPV4, appears to act as a molecular integrator of converging signals. Such a range of signaling diversity has not been demonstrated in other vanilloid channel family members (TRPV2, TRPV3, TRPV5, and TRPV6), but many of their properties and gating behaviors may still remain to be discovered (4, 9).
Endogenous TRPV4 Distribution and FunctionAlthough the gating properties of the exogenously expressed TRPV4 channel are beginning to be elucidated in heterologous expression systems, only limited data are available on the functional important of the endogenous TRPV4 channel, although accumulating evidence demonstrating the tissue and cellular distribution patterns of the channel may provide insights into channel function. To date, the channel has been shown to be expressed in a wide range of tissues and cells, results consistent with a "broad" importance in biology. The channel has been identified at high levels in trachea and kidney but also is expressed in liver, heart, lung, testis, brain, sensory ganglia, inner/outer ear, salivary gland, pancreas, and fat (15, 16, 17, 18). At the cellular level, TRPV4 has been identified in a diverse range of cells, including various central nervous system nerve fibers/organs and sympathetic ganglia (15, 16), auditory ganglia and hair cells of inner/outer ear (16), renal glomerular and distal tubule epithelial cells (15, 17), epithelial cells lining trachea and lung airway (15), skin keratinocytes (22), and aortic endothelial cells (18). In our own studies of kidney cells, we have identified TRPV4 mRNA in various renal cell cultures, including mouse mesangial cells (smooth muscle-like) and several epithelial cells (MCT-proximal tubule cells, M-1 cortical collecting duct cells, and IMCD-3 inner medullary collecting duct cells).2 Further, in C. elegans, OSM-9 has been identified in numerous sensory neurons. Hence, the channel is widely distributed in neuronal cells, epithelial cells, some muscle cells, and in at least one endothelial cell.
Although the distribution of the TRPV4 channel has been shown to be
diverse, as heretofore noted, the function of the endogenous channel in a
specific environment and cell type has only been partially identified. In
mouse aortic endothelial cells, which express TRPV4, both hypotonic swelling
and application of 4-PDD can activate an apparent endogenous TRPV4
channel (21). We have shown
that in mouse kidney M-1 epithelial cells (cortical collecting duct cells),
both hypotonic swelling and shear stress (or flow rate/pressure) can activate
an endogenous TRPV4 channel, based on sensitivity to ruthenium
red.2 A swelling- and
stretch-activated calcium-permeable channel was previously identified in
kidney proximal tubule cells
(35,
36), but it appears to differ
from TRPV4 in that it requires stress-induced translocation of PKC to the
plasma membrane for activation
(25). Nonetheless, because
renal tubule cells can experience a wide range of hypotonic and shear stress
(or flow rate/pressure) conditions, leading to increased influx of calcium in
both proximal tubule cells
(26,
27,
35,
36) and late distal
tubule/collecting duct cells
(3741),
it is likely that the endogenous TRPV4 channel expressed in renal cells will
be regulated by these physical stresses. In addition, both 4
-PDD and
PMA at low concentration (100 nM) can lead to activation of the
endogenous TRPV4 channel in M-1
cells.2 Because
PKC-dependent signals can activate TRPV4, specific hormones known to operate
through PKC and to activate calcium influx in cortical collecting duct cells,
such as endothelin 1 and vasopressin
(4244),
may be exerting their effects via PKC-dependent activation of the TRPV4
channel.
In summary, it has been shown that temperature is a critical modulator of TRPV4 channel gating, leading to activation of the channel by a diverse range of microenvironmental chemical and physical signals. This diversity of activating signals is transduced through at least two transduction pathways, one PKC-dependent and one PKC-independent, although other possible pathways are likely to be discovered. Because multiple signals and transduction pathways converge on the same channel, the channel would appear to function as a molecular integrator of microenvironmental chemical and physical signals.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Integrative Biology and
Pharmacology, The University of Texas Health Science Center at Houston, 6431
Fannin, Houston, TX 77030. E-mail:
roger.g.oneil{at}uth.tmc.edu.
1 The abbreviations used are: 4-PDD, 4
-phorbol
12,13-didecanoate; Cal C, calphostin C; ISO, isotonic medium; HYPO, hypotonic
medium; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RR,
ruthenium red; SS, shear stress; CHO, Chinese hamster ovary; HEK, human
embryonic kidney; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered
saline; TRP, transient receptor potential.
2 X. Gao and R. G. O'Neil, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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