Temperature-modulated Diversity of TRPV4 Channel Gating

ACTIVATION BY PHYSICAL STRESSES AND PHORBOL ESTER DERIVATIVES THROUGH PROTEIN KINASE C-DEPENDENT AND -INDEPENDENT PATHWAYS*

Xiaochong Gao, Ling Wu and Roger G. O'Neil {ddagger}

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells resulting in high levels of expression at the plasma membrane. Channel activation was assessed from changes in calcium influx (fura-2 fluorescence measurements) or whole cell currents (patch clamp analysis). At room temperature (22–24 °C), exposure of TRPV4-transfected cells to hypotonic medium (225 mOsm/liter) or a non-protein kinase C (PKC)-activating phorbol ester derivative, 4{alpha}-phorbol 12,13-decanoate (100 nM), induces modest channel activation, whereas phorbol 12-myristate 13-acetate (100 nM), a PKC-activating phorbol ester, and shear stress (3–20 dyne/cm2) had minimal or no effect on channel activation. In contrast, at elevated temperatures (37 °C) the channel was rapidly activated by all stimuli. Inhibition of PKC by calphostin C (50 nM) or staurosporine (500 nM) abolished phorbol 12-myristate 13-acetate-induced activation of the channel without affecting the response to other stimuli. Ruthenium red (1 µM) effectively blocked the channel activity by all stimuli. It is concluded 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 utilizing a least two transduction pathways, one PKC-dependent and one PKC-independent. The convergence of multiple signals and transduction pathways on the same channel indicate that the channel functions as a molecular integrator of microenvironmental chemical and physical signals.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The TRP calcium-permeable cation channels are a rapidly growing superfamily of channels expressed in a broad range of both excitable and nonexcitable cells. The channels, in general, are not voltage-activated but appear to be activated by a broad range of ligands and intracellular mediators (17). The superfamily has been divided into three main subfamilies based on structural and functional similarities: TPRVC, the canonical TRP channels first identified in Drosophila; TRPV, the vanilloid subfamily named after its first member, the vanilloid receptor (or capsaicin receptor; renamed TRPV1); and TRPM, the melatonin subfamily named after its first member, melatonin (see Refs. 1 and 8 for nomenclature). The TRPV subfamily has demonstrated a notable broad sensitivity to chemical and physical stimuli, particularly for TRPV1, which is activated by various noxious mediators, including heat, acid, and capsaicin (the hot ingredient of peppers), and by endogenous lipid mediators, such as diacylglycerol and anandamide (2, 5, 9). Other members of the TRPV subfamily have been shown, so far, to have a narrower range of sensitivities: heat and insulin-like growth factor-I for TRPV2 (10, 11); heat for TRPV3 (1214); and vitamin D and related ligands for TRPV5 (4).

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{alpha}-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 22–24 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).


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of the TRPV4 Channel—The full-length TRPV4 cDNA coding region was obtained using homology-based reverse transcription-PCR amplification procedures and designed primer sets (SuperScript III First Strand Synthesis System for reverse transcription-PCR; Invitrogen) from mRNA isolated (RNeasy kit; Qiagen) from mouse M-1 cells (American Type Tissue Culture). The full-length cDNA was ligated into the pcDNA3.1/V5-His vector (Invitrogen, Inc.) and fused with a 14-amino acid V5 epitope on the C-terminal end of the TRPV4 cDNA using the TOPOTM cloning technique (pcDNA3.1/V5-His TOPOTM TA expression kit; Invitrogen) to provide the pcDNA3.1/TRPV4-V5 plasmid. The recombinant pcDNA3.1/TRPV4-V5 plasmid was transformed into chemically competent TOP10 Escherichia coli cells using standard chemical transformation procedures. Up to 10 positive colonies were selected, and the plasmid DNA was isolated using the QIAprep® Spin miniprep kit (Qiagen). The plasmid DNA was analyzed by sequencing with designed sequencing primers (sequenced by SeqWright, Inc.) to verify the TRPV4 cDNA insert size and orientation. The full-length sequence was identical to that previously reported (17, 18).

Cell Culture and TRPV4 Expression—HEK 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{alpha} 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 48–72 h, ~30–60% 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{alpha}-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{alpha}-PDD), likewise demonstrated cells expressing the channel.

Immunohistochemistry and Immunoblotting—Immunohistochemical 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 (5–10 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 Calcium—The 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 5–10 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 10–15 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 10–30 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 1–3 s. The fura-2 fluorescence ratios were converted to intracellular calcium activity ([Ca2+]i) as described by Grynkiewicz et al. (28).

(Eq. 1)
where R is the ratio at any time and {beta} is the ratio of the fluorescence emission intensity at 380 nm excitation in Ca2+-depleting and Ca2+-saturating conditions. Kd is the Ca2+ dissociation constant of fura-2 (Kd = 220 nM). Rmin is the minimum ratio in Ca2+-depleting conditions (addition of 5 mM EGTA and 2 µM ionomycin), and Rmax is the maximum ratio in Ca2+-saturating conditions (2 µM ionomycin plus 5 mM Ca2+) determined under the conditions of the experiment.

Shear Stress—For 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 (0–75 ml/min) to generate shear stresses from 0 to 20 dyne/cm2 while simultaneously monitoring intracellular calcium levels in individual cells (typically 10–30 cells), and the results were averaged for each coverslip.

Patch Clamp Current Measurements—The 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 2–4M{Omega} (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 40–80%. 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 Chemicals—HEK 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 {alpha}-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 1–10 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.

Statistics—Intracellular 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of TRPV4—Expression of the TRPV4 protein in HEK 293 cells and CHO cells was readily detected with the anti-V5-FITC antibody. As shown Fig. 1 (A and D), in TRPV4-transfected cells (T-HEK and T-CHO), TRPV4 protein expression was readily detected within the cells. The channel was shown to be highly expressed in the perinuclear region of both the HEK 293 and CHO cells, reflecting expression in the endoplasmic reticulum and Golgi apparatus and in the plasma membrane. As evident in the figure, detection of protein expression via the V5 epitope construct in the present study permitted excellent assessment of protein localization at the plasma membrane. In the absence of transfection (HEK and CHO), binding of the anti-V5-FITC antibody was not evident, demonstrating little, or an absence of, expression of the TRPV4-V5 construct. The observed pattern of expression is consistent with the results noted by others where a TRPV4 construct was employed utilizing green fluorescent protein, a relatively large protein, attached to the C terminus as a fluorescent tag (17, 18).



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FIG. 1.
Immunocytochemistry and immunoblotting of the TRPV4 construct expressed in HEK 293 cells and CHO cells. Immunocytochemical localization of TRPV4 expression in TRPV4-transfected HEK 293 cells (A, T-HEK) and CHO (D, T-CHO) cells was visualized with the anti-V5-FITC antibody. Strong expression was apparent at the plasma membrane and perinuclear regions (see text for details). TRPV4 expression was not apparent in nontransfected cells (B, HEK; E, CHO). TRPV4 expression was also detected by immunoblotting (detected with anti-V5-HRP) in TRPV4-transfected HEK 293 (C, T-HEK) and CHO (F, T-CHO) cells but not in nontransfected cells (C, HEK; F, CHO). The detected single band in transfected cells had a molecular mass of ~100 kDa (arrow) that includes the C-terminal fusion segment containing the V5-epitope of 5 kDa.

 

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 Levels—The 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 (22–24 °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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FIG. 2.
Effect of temperature on the steady-state basal [Ca2+]i levels in TRPV4-transfected and nontransfected cells. A, the effect of incubating HEK 293 cells at room temperature (RT) or 37 °C (37 °C) on basal [Ca2+]i levels in nontransfected (HEK) and TRPV4-transfected (T-HEK) cells (n = 9, 9, 9, and 9, respectively). B, the effect of incubating CHO cells at room temperature (RT) and 37 °C (37 °C) on basal [Ca2+]i levels in nontransfected (CHO) and TRPV4-transfected (T-CHO) cells (n = 10, 8, 10, and 10, respectively). *, p <= 0.5 compared with control room temperature values.

 

Temperature-modulated Gating of TRPV4 by Phorbol Esters—It has been shown previously that phorbol ester derivatives, such as PMA and 4{alpha}-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{alpha}-PDD are PKC-independent. Hence, the effects of temperature on the actions of both PMA and 4{alpha}-PDD at 100 nM were assessed.

In studies at room temperature in transfected HEK 293 cells, the application of 4{alpha}-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{alpha}-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{alpha}-PDD was observed in nontransfected HEK 293 cells (data not shown).



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FIG. 3.
Effect of temperature on TRPV4 activation in TRPV4-transfected HEK 293 cells. The effect of application of the signaling stimulants was assessed at room temperature (RT) and at 37 °C as indicated in the figure. A, representative examples showing the effect of 4{alpha}-PDD (100 nM) on [Ca2+]i at room temperature and 37 °C. B, summary of 4{alpha}-PDD-induced change in [Ca2+]i, {Delta}[Ca2+]i (n = 5 and 5, respectively). C, representative examples showing the effect of PMA (100 nM) on [Ca2+]i at room temperature and 37 °C. D, summary of PMA-induced change in [Ca2+]i, {Delta}[Ca2+]i (n = 3 and 6, respectively). E, representative examples showing the effect of HYPO on [Ca2+]i at room temperature and 37 °C. F, summary of HYPO-induced change in [Ca2+]i, {Delta}[Ca2+]i (n = 6 and 6, respectively). G, representative examples showing the effect of SS (10 dyne/cm2)on[Ca2+]i at room temperature and 37 °C. H, summary of SS-induced change in [Ca2+]i, {Delta}[Ca2+]i (n = 4 and 4, respectively). Inset, SS-induced {Delta}[Ca2+]i for SS of 3, 10, and 20 dyne/cm2 at 37 °C (n = 3, correlation coefficient = 0.920, p < 0.001). *, p <= 0.05 compared with control room temperature values.

 

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 Stresses—Physical 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 Gating—Some 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{alpha}-PDD or PMA (Fig. 4, A–D). 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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FIG. 4.
Effect of extracellular calcium and PKC inhibition on TRPV4 activation in TRPV4-transfected HEK 293 cells. The effect of application of the signaling stimulants was assessed at 37 °C in the presence or absence of extracellular calcium (+Ca and -Ca, respectively), or following inhibition of PKC by addition of 50 nM calphostin C (+Cal C). Cal C was added 10 min prior to activation of the channel. A, representative time courses of [Ca2+]i following addition of 4{alpha}-PDD (100 nM) in the presence of calcium (+Ca), the absence of calcium (-Ca), or following addition of Cal C (+Ca, +Cal C). B, summary of 4{alpha}-PPD-induced changes in [Ca2+]i, {Delta}[Ca2+]i (n = 6, 4, and 6, respectively). C, representative time courses of [Ca2+]i following addition of PMA (100 nM) in the presence of calcium (+Ca), absence of calcium (-Ca), or following addition of Cal C (+Ca, +Cal C). D, summary of PMA-induced changes in [Ca2+]i, {Delta}[Ca2+]i (n = 11, 2, and 11, respectively). E, representative time courses of [Ca2+]i following application of HYPO in the presence of calcium (+Ca), absence of calcium (-Ca), or following addition of Cal C (+Ca, +Cal C). F, summary of HYPO-induced changes in [Ca2+]i, {Delta}[Ca2+]i (n = 5, 3, and 5, respectively). *, p <= 0.05 compared with control values.

 

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{alpha}-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{alpha}-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{alpha}-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 TRPV4—To 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 (1–10 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{alpha}-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{alpha}-PDD, PMA, HYPO, and shear stress by 96, 92, 88, and 68%, respectively (Fig. 5, B–E). 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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FIG. 5.
Effect of RR inhibition on TRPV4 activation in TRPV4-transfected HEK 293 cells. The effect of application of signaling stimulants was assessed at 37 °C in the presence of RR (1 µM). RR was normally added just prior to the stimulus (1 min) except for SS when it was added during the stimulus. A, representative example showing the effect of addition of RR on the 4{alpha}-PPD-induced change in [Ca2+]i, {Delta}[Ca2+]i. Similar results were observed for the other stimulants (examples not shown). B, summary of the effect of addition of 4{alpha}-PDD on {Delta}[Ca2+]i in the absence of RR (+Ca) and presence of RR (+Ca, +RR) (n = 4 and 4, respectively). C, summary of the effect of addition of PMA on {Delta}[Ca2+]i in the absence of RR (+Ca) and presence of RR (+Ca, +RR) (n = 4 and 4, respectively). D, summary of the effect of application of HYPO on {Delta}[Ca2+]i in the absence of RR (+Ca) and presence of RR (+Ca, +R) (n = 4 and 4, respectively). E, summary of the effect of application of SS on {Delta}[Ca2+]i in the absence of RR (+Ca) and presence of RR (+Ca, +RR) (n = 4 and 4, respectively). *, p <= 0.05 compared with control values.

 

Patch Clamp Analysis of TRPV4 Currents—To 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{alpha}-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{alpha}-PDD at 37 °C. In nontransfected cells, neither 4{alpha}-PPD nor PMA produced an increase in whole currents as shown in Fig. 6 (A and B), respectively. In contrast, both 4{alpha}-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{alpha}-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{alpha}-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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FIG. 6.
Whole cell currents (I) in TRPV4-transfected (T-HEK) and nontransfected HEK (HEK) cells at 37 °C. All of the currents were recorded at a holding potential of -60 mV (cell interior negative) unless otherwise noted. A, representative example of 4{alpha}-PDD (100 nM) actions on I of nontransfected HEK cells. B, representative example of PMA (100 nM) actions on I of nontransfected HEK cells. C, representative example of 4{alpha}-PDD (100 nM) actions on I of TRPV4-transfected HEK cells. 4{alpha}-PDD induces a strong inward current (negative current). D, representative example of PMA (100 nM) actions on I of TRPV4-transfected HEK cells. PMA induces a strong inward current (negative current). E, I-V relation of TRPV4-transfected HEK cells before (Ctl) and after 4{alpha}-PDD addition (4{alpha}-PDD). Note the outward rectification of current with positive membrane voltages (positive I reflects outward currents with positive membrane voltages; negative I reflects inward currents with negative membrane voltages). F, I-V relation of TRPV4-transfected HEK cells before (Ctl) and after PMA addition (PMA). Note the outward rectification of current with positive membrane voltages. G, summary of the peak changes in I (I) upon addition of 4{alpha}-PDD (4{alpha}PDD) or PMA (PMA) in TRPV4-transfected cells (n = 5).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Temperature-modulated Diversity of Gating Signals—The mammalian TRPV4 channel is a calcium-permeable channel that has been shown previously to be activated by osmotic stress and phorbol esters derivatives through non-PKC-dependent pathways when studied at room temperature (16, 17, 18, 21). This limited range of gating signals is in contrast to that thought to exist for the TRPV4 homologue in C. elegans, OSM-9, where accumulating evidence points to a central role of OSM-9 in a broad range of sensory functions, including olfaction, mechanosensation, chemosensation, and osmosensation, results consistent with the presence of a broad range of gating signals or polymodal gating behavior (19, 20). The present study demonstrates that elevated temperatures near body temperature appear to critically modulate, or potentiate, the diversity of signals that are linked to activation of the mammalian channel. At 37 °C, the channel was potently regulated by hypoosmotic stress (swelling), mechanical stress (shear stress), and chemical signals (phorbol ester derivatives) through both PKC-dependent pathways and PKC-independent pathways. Others have shown that temperature itself may activate the channel, at least transiently, in the non-noxious temperature range (30–43 °C) (22, 23), further demonstrating the diverse gating behavior of the mammalian channel. It may be that elevation of temperature above body temperature may unmask further regulatory diversity of the channel not previously recognized.

A Molecular Integrator of Microenvironmental Signals—The 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 Function—Although 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{alpha}-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{alpha}-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.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK 40545 (to R. G. O.) and American Heart Association (Texas Affiliate) Grant-in-Aid 0150758Y (to R. G. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} 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{alpha}-PDD, 4{alpha}-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. Back

2 X. Gao and R. G. O'Neil, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Drs. Andy Morris and Rachel Brown for critical reading of the manuscript.



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 ABSTRACT
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