Versatile Regulation of Cytosolic Ca2+ by Vanilloid Receptor I in Rat Dorsal Root Ganglion Neurons*

Min LiuDagger , Meng-Chuan LiuDagger , Charalambos Magoulas§, John V. PriestleyDagger , and Nick J. WillmottDagger

From the Departments of Dagger  Neuroscience and § Neurosurgery, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Queen Mary University of London, Medical Sciences Building, Mile End Road, London EC1 4NS, United Kingdom

Received for publication, September 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca2+. The VR1 agonist capsaicin induced Ca2+ mobilization from intracellular stores in the absence of extracellular Ca2+, and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca2+ mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca2+ release receptor. The main part of the capsaicin-releasable Ca2+ store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca2+-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca2+ store. In addition, VR1 was observed to behave as a store-operated Ca2+ influx channel. In DRG neurons, capsazepine attenuated Ca2+ influx following thapsigargin-induced Ca2+ store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca2+ influx and inward currents following Ca2+ store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca2+ with the plasma membrane-associated form behaving as a store-operated Ca2+ influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca2+ release receptor in sensory neurons.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vanilloid receptor 1 (VR11 or TRPV1) belongs to the transient receptor potential (TRP) family of nonspecific cation channels and has been proposed to be analogous to the capsaicin receptor of sensory neurons (1). VR1 confers several sensory functions in these cells, including the transduction of chemical (vanilloids and pH) and physical (heat) stimuli resulting in the generation of action potentials in nociceptive nerve endings, which are ultimately responsible for the sensations of heat and thermal/inflammatory pain (2, 3). Although VR1 exhibits Ca2+-dependent desensitization and has been proposed to be modulated by protein kinase C (4, 5), it is possible that this receptor is subject to many other regulatory mechanisms that may be of fundamental importance to its functioning in sensory neurons. Although the activation of certain members of the TRP family of cation channels is reliant on the state of filling of intracellular Ca2+ stores (6), with these channels being activated by intracellular Ca2+ store depletion, it remains controversial whether VR1 has an innate capacity to function as a store-operated Ca2+ channel (SOCC) in sensory neurons. To date, there is no published evidence to support this, a previous study failing to detect activation of expressed VR1 following Ca2+ store depletion by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin in Xenopus oocytes (1).

In addition to functioning as a plasma membrane cation influx channel, VR1 may also behave as a Ca2+ release receptor. It has recently been reported that the activation of a capsaicin receptor induces the release of Ca2+ from ryanodine receptor-linked intracellular Ca2+ stores of rat dorsal root ganglion (DRG) neurons in the absence of extracellular Ca2+ (7). Additionally, VR1 expressed in HEK-293 cells colocalizes to endoplasmic reticulum (ER), and vanilloids induce Ca2+ release from intracellular stores in these transfected cells (8). However, it remains to be established whether VR1 is a functional component of ER in sensory neurons, which would be mandatory for the classification of this protein as a Ca2+ release receptor in these cells. Considering the above, it is possible that functional diversity for VR1 might account for the large and sustained increase in cytosolic Ca2+ ([Ca2+]i) observed in DRG neurons subjected to long term, high vanilloid concentration exposure. It is well established that this treatment results in profound ultrastructural changes in DRG neurons (9, 10) and cell death (11, 12), although there is scant evidence of a causal link between cell pathology and a vanilloid-induced sustained elevation of [Ca2+]i involving detrimental Ca2+ release from the ER in these cells.

To reconcile whether VR1 is functionally multifaceted in its regulation of [Ca2+]i, this study endeavored to determine whether VR1 might act as a SOCC and a Ca2+ release receptor in DRG neurons. Data obtained from Ca2+ imaging, confocal microscopy, and electrophysiological experiments suggest that ER-associated VR1 may be predominantly localized to a thapsigargin-insensitive Ca2+ store in DRG neurons, which can be readily depleted by capsaicin receptor binding, and that plasma membrane VR1 is activated by Ca2+ store depletion in DRG neurons and transfected HEK-293 cells. These data therefore present a case for plasma membrane VR1 functioning as a SOCC and ER-associated VR1 functioning as a Ca2+ release receptor in DRG neurons.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DRG Neuron Culture-- DRG neurons were prepared from adult male Sprague-Dawley rats (150-200 g) by methods similar to those described previously (13). Isolated ganglia were incubated with 2 ml of modified Bottenstein's culture medium (BSF-2) consisting of 0.3% bovine serum albumin, 1% N-2 supplement, and 0.125% collagenase XI in Ham's F12 basal medium at 37 °C for 2 h and were triturated with a glass pipette to homogeneity. Dissociated cells were centrifuged at 500 × g for 5 min through a cushion of Hanks' buffered salt solution (HBSS) containing 15% (w/v) bovine serum albumin. The dispersed cells were resuspended in Ham's F-12 basal medium containing 100 ng/ml nerve growth factor 7 S, 0.3% bovine serum albumin, 1% N-2 supplement, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were plated in 35-mm culture dishes (Corning Glass, Corning, NY) containing 0 thickness, 22-mm-diameter coverslips (BDH Chemicals) coated previously with 50 µg/ml poly-L-lysine. Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 1-2 days prior to use.

HEK-293 Cell Transfection and Culture-- HEK-293 cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) as a frozen culture. Cells were thawed and then grown in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Cells were subcultured every third day and grown to 80-90% confluence. Cells were detached from plates using 0.05% trypsin-EDTA solution and subsequently neutralized by addition of 10 volumes of the above culture medium. Stock cell preparations were stored in 10% glycerol in liquid nitrogen until required for experiments.

Transfection of HEK-293 cells (grown in 35-mm Petri dishes to ~80% confluence) was carried out using LipofectAMINE 2000 (Invitrogen) as described by the manufacturer. For each dish, 1 µg of VR1 plasmid (pcDNA3.1/rVR1, a generous gift from Dr. Xuenong Bo, Sheffield University, Sheffield, UK) together with 0.3 µg of pEGFP (Clontech) were mixed with diluted lipid reagent (4 µl in 100 µl of Optimem 1) for 15 min at 22 °C. Cells were incubated with the above complexes in 1 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air and 5% CO2, and after 5 h, the volume of medium was increased to 2 ml. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 1-4 days prior to use.

Labeling DRG Neurons with Fluorescent Probes to ER and VR1-- DRG neurons were incubated with either 0.6 µM ER-tracker blue-white dapoxyl (Molecular Probes Inc) or 0.1 µM of the carbocyanine ER probe DiOC5 in HBSS (pH 7.2) for 20 min at 22 °C. Coverslips containing DRG neurons were washed in 3 × 5 ml aliquots of HBSS for 10 min and were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 15 min at 22 °C. Coverslips were washed in 3 × 5 ml aliquots of PBS for 10 min and were incubated with 10% donkey serum at 22 °C for 1 h to block nonspecific binding sites. Cells were incubated with guinea pig polyclonal antibody to VR1 (1:400, diluted in antibody buffer comprising of 0.2% Triton X-100 and 0.1% sodium azide in PBS) for 2 h, washed in 3 × 5 ml aliquots of PBS for 10 min, and then incubated with TRITC (tetramethyl rhodamine isothiocyanate)-conjugated donkey anti-guinea pig IgG (1:400) for 1 h. Following three final washes with PBS for 10 min, cultures were mounted in PBS glycerol supplemented with DABCO (1,4-diazobicyclo-(2, 2, 2)-octane).

Confocal Microscopy-- Small sized DRG neurons (15-30-µm diameter) labeled with fluorescent probes for ER and VR1 (as above) were examined using an upright Zeiss confocal laser scanning microscope system (LSM 510) and a ×40 Plan Neofluor oil objective. To study the distribution and localization of intracellular fluorescence, cells were scanned in multitracking mode (to avoid channel cross talk) for TRITC (Ex = 543 nm, Em = 580 nm) and either ER-tracker blue-white dapoxyl (Ex = 351 nm, Em = 460 nm) or DiOC5 (Ex = 488 nm, Em = 515 nm). Increments in the Z plane were 0.2 µm with confocal images being acquired throughout the central portion of cells.

Ratiometric Measurement of [Ca2+]i-- Capsaicin-induced increases in [Ca2+]i were assessed in the soma of small sized (15-30-µm diameter) rat DRG neurons cultured for 1-2 days by a similar method as described previously (14). Cells were loaded with Ca2+ indicator by incubation with HBSS containing 2 µM of the acetoxymethyl ester of fura-2 for 2 h. Intracellular fluorescence was imaged at 22 °C in a nominally Ca2+-free Hanks' buffered salt solution with no added Ca2+ and containing 0.5 mM EGTA (pH 7.2). The equipment used included an inverted microscope (Nikon Diaphot) with a ×40 Plan Fluor oil-immersion objective (1.3 NA), coupled to a xenon arc lamp/fast filter switching device (Sutter DG4: Sutter Instruments, Novato, CA), and 12-bit interline CCD (Sensicam, PCO Computer Optics, Kelheim, Germany), which were both interfaced to a Pentium III computer. A low temperature (22 °C) was used for Ca2+ imaging experiments as fura-2 readily compartmentalizes into cellular organelles at higher, physiological temperatures in these cell preparations. All cultures were maintained in a static, open chamber throughout experiments. Free cytosolic Ca2+ was quantified by taking the ratio of fluorescence intensities at excitation wavelengths 340 and 380 nm, using an emission wavelength of 510 nm. Pairs of 340- and 380-nm images were captured every 8.0 s, and ratio images were calculated using Axon Imaging Workbench 4.0 software (Axon Instruments, Foster City, CA). Standard CaCl2 solutions were used to calibrate the system for measurements with fura-2, and viscosity corrections were made. For comparative pharmacological experiments, naive and drug-treated DRG neurons were imaged on the same day, using the same batch preparation of cells. Response parameters (Delta [Ca2+]i) were compared for statistical significance using Student's t test for unpaired observations.

Electrophysiology-- Whole-cell voltage clamp recording was carried out at 22 °C using an Axopatch-1D amplifier (Axon Instruments). Patch pipettes were typically of 2-5 megaohms resistance when filled with solution containing 140 mM KCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.3). In some experiments, K+ was replaced by Cs+ at the same concentration in the pipette solution. Cell series resistance was compensated to 40-80%. The membrane was voltage-clamped at -70 mV. Signals were filtered at 1 kHz and sampled at 1 kHz using WinWCP V3.08 software. The recording chamber was perfused continuously (by gravity) with extracellular bathing solution at a flow rate of 5 ml/min and contained 135 mM NaCl, 5 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES (pH 7.4). Drugs were applied to small sized rat DRG neurons (15-30 µm diameter) or transfected HEK-293 cells focally (by gravity) via a large bore pipette with a tip diameter of 100 µm, positioned within 200 µm of the cell body. Sustained inward currents induced by 1 µM thapsigargin (Tg) were continuously recorded. Capsazepine (1 µM) was applied to Tg-pretreated cells shortly after inward currents induced by Tg reached a plateau. Capsaicin sensitivity was tested in DRG neurons and transfected HEK-293 cells by measuring inward currents elicited by the addition of 1 µM capsaicin to cells. The amplitudes of inward currents were compared for statistical significance using Student's t test for unpaired observations.

Drugs and Solutions-- HBSS (pH 7.2) contained 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 0.83 mM MgSO4, 0.42 mM Na2HPO4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 5 mM glucose. For Ca2+ imaging experiments carried out in nominally Ca2+-free medium, CaCl2 was omitted from the above, and 0.5 mM EGTA was added, yielding a free Ca2+ concentration of ~10 nM. PBS (pH 7.4) contained 0.01 M sodium phosphate and 0.14 M NaCl. Dulbecco's modified Eagle's medium, fetal bovine serum, Ham's F-12 basal medium, N-2 supplement, bovine serum albumin, penicillin, streptomycin, LipofectAMINE 2000, and Optimem 1 were from Invitrogen. Guinea pig polyclonal antibody to VR1 was from Neuromics Inc. (Minneapolis, MN). TRITC-conjugated donkey anti-guinea pig IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). GFP plasmids (pEGFP) were from Clontech, and VR1 plasmids (pcDNA3.1/rVR1) were a generous gift from Dr. Xuenong Bo, Sheffield University. Fura-2 AM, DiOC5, and ER-tracker blue-white dapoxyl were from Molecular Probes Inc. Thapsigargin, ruthenium red, and xestospongins were from Calbiochem. All other drugs and reagents were from Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lipid soluble VR1 agonist capsaicin and the ER Ca2+-ATPase inhibitor Tg mobilized Ca2+ from intracellular stores of small sized DRG neurons (15-30 µm diameter) in the absence of extracellular Ca2+ (Fig. 1). From the examination of dose-response curves, it was evident that 1 µM of either of these agents induced a maximal Ca2+ release (Delta [Ca2+]i) in cells, using our static incubation protocol (Fig. 1). Moreover, second bolus applications of Tg or capsaicin resulted in no further Ca2+ release in cells (Fig. 2a), suggesting that 1 µM capsaicin or Tg induced a maximal depletion of the respective Ca2+ stores, which were sensitive to these agents. Although Tg (1 µM) induced Ca2+ mobilization in glial cells (which could be clearly discriminated from phase-bright DRG neurons in cultures), capsaicin (1 µM) failed to increase [Ca2+]i in the presence or absence of extracellular Ca2+ in glia (data not shown), indicating that capsaicin-induced responses were sensory neuron-specific. The Ca2+ ionophore ionomycin induced Ca2+ mobilization in Tg- and capsaicin-pretreated DRG neurons, indicating the presence of a releasable pool of Ca2+, which was insensitive to these agents (Fig. 2a), whereas pretreatment of cells with ionomycin resulted in an anticipated elimination of both Tg- and capsaicin-sensitive Ca2+ stores (Fig. 2b). The VR1 antagonist capsazepine (10 µM) induced a slow release of Ca2+ from intracellular stores and abrogated the capsaicin-induced transient increase in [Ca2+]i (Fig. 2c), suggesting that capsaicin-induced Ca2+ release was dependent on capsaicin receptor binding and that capsazepine probably acts as a partial agonist for this receptor-mediated Ca2+ release. Capsaicin-induced Ca2+ release was unaffected by the phospholipase C inhibitor xestospongins (Fig. 2d) indicating that intracellular inositol-1,4,5-trisphosphate generation was not responsible for the observed Ca2+ mobilization.


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Fig. 1.   Capsaicin evoked Ca2+ release from intracellular stores of small sized DRG neurons in the absence of extracellular Ca2+. Each left-hand trace in this figure is a population mean of the [Ca2+]i of at least seven cells from a single coverslip, and all mean traces are representative of at least three separate experiments. Dose-response curves representing the maximum change in [Ca2+]i (Delta [Ca2+]i) versus [capsaicin] (a) or [Tg] (b) are shown (right-hand graphs) with each data point representing the mean Delta [Ca2+]i ± S.E. of at least 15 cells from three separate experiments. Data were fitted to a sigmoid curve using a Boltzman function, and EC50 ± S.D. and maximum response (Rmax) ± S.D. values derived from the curves of best fit are indicated in each graph.


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Fig. 2.   Capsaicin-induced Ca2+ release from intracellular stores of small sized DRG neurons is reliant on capsaicin-receptor binding and not IP3 generation. Each trace in this figure is a population mean ± S.D. of the [Ca2+]i of at least 10 cells from a single coverslip, which demonstrated Ca2+ mobilization in the absence of extracellular Ca2+, and all traces are representative of at least three separate experiments. As shown in a, 1 µM capsaicin or 1 µM Tg induced a maximal depletion of the respective Ca2+ stores that were sensitive to these agents with second applications of Tg or capsaicin eliciting no further Ca2+ release. Ionomycin released Ca2+ from remaining stores, indicating the presence of a releasable pool of Ca2+ that was insensitive to capsaicin or Tg. As shown in b, both Tg- and capsaicin-sensitive Ca2+ stores of DRG neurons were depleted by ionomycin. As shown in c, addition of 10 µM capsazepine to the bath resulted in a slow release of Ca2+ from intracellular stores and the abrogation of the capsaicin-induced transient increase in [Ca2+]i. As shown in d, capsaicin-induced Ca2+ release was unaffected by the phospholipase C inhibitor xestospongins (10 µM).

Confocal microscopy revealed heterogeneous staining patterns for the TRITC-based VR1 probe in small sized DRG neurons. Although 74% of cells (31 out of 42 cells analyzed) demonstrated varying levels of plasma membrane and intracellular staining, the remainder did not stain significantly with the VR1 probe, suggesting negligible VR1 expression in these (Fig. 3a). Evidence that intracellular VR1 is associated with the ER in a large proportion of small sized DRG neurons was derived from double-labeling experiments. In Fig. 3, b and c, the far right images are overlays of the left and middle images and highlight colocalization for ER and VR1 probes (yellow) to the same intracellular organelles in small sized DRG neurons. Approximately 70% of small sized cells (20 out of 28 cells analyzed) demonstrated similar intracellular colocalization for ER and VR1 as the cells of Fig. 3, b and c. In the remainder, intracellular VR1 staining was negligible, consistent with the single probe experiments of Fig. 3a.


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Fig. 3.   Intracellular VR1 is extensively localized to the ER in small sized DRG neurons. a, red pseudocolor fluorescence image showing typical heterogeneous staining patterns of the TRITC-based VR1 probe in five small sized DRG neurons. Staining to the plasma membrane is evident in cell 1, whereas considerable intracellular punctate staining is present in cell 2 and to a far lesser extent in cells 3 and 4. There is negligible VR1 staining in cell 5. b, DRG neurons labeled with the fluorescent ER probe DiOC5 and the probe to VR1. Confocal microscopy of double-labeled cells revealed extensive intracellular colocalization for VR1 and ER in small sized DRG neurons. The far left (pseudocolored red) and middle (pseudocolored green) images show intracellular fluorescence of the VR1 probe and the ER probe respectively, in three small sized cells. The far right image is an overlay of the left and middle images and highlights colocalization for ER and VR1 probes (yellow) to the same intracellular organelles. As shown in c, essentially identical staining patterns were observed substituting DiOC5 for the specific ER probe, ER-tracker blue-white dapoxyl (middle image, pseudocolored green) in double-labeling experiments. As for panel b, the far right image in panel c is an overlay of the images representing VR1 and ER staining (left and middle images). Scale bars in a, b, and c = 10 µm.

A further examination of Tg- and capsaicin-sensitive Ca2+ stores revealed that only a subset of these pharmacologically overlap in small sized DRG neurons (Fig. 4). Mobilization of Ca2+ from intracellular stores was assessed by measuring the mean maximum change in [Ca2+]i (Delta [Ca2+]i) in response to a sequential challenge of Tg, capsaicin, and ionomycin (Fig. 4a) or capsaicin, Tg, and ionomycin (Fig. 4c). From these experiments, four pharmacologically distinct Ca2+ stores were identified in small sized cells. These included capsaicin-sensitive/Tg-insensitive (Fig. 4a, middle response of the trace), Tg-sensitive/capsaicin-insensitive (Fig. 4c, middle response of the trace), capsaicin-insensitive/Tg-insensitive/ionomycin-sensitive (Fig. 4, a and c, final responses of the traces), and capsaicin-sensitive/Tg-sensitive (inferred from Fig. 4e) Ca2+ stores. Although nearly all (>95%) small sized DRG neurons analyzed possessed a Tg-sensitive or a Tg-sensitive/capsaicin-insensitive store, only 74 and 65% of cells possessed a capsaicin-sensitive or a capsaicin-sensitive/Tg-insensitive Ca2+ store, respectively (Fig. 4d). It is therefore notable that a similar proportion of small sized DRG neurons possessed capsaicin-sensitive Ca2+ stores (74%) as the proportion of cells that demonstrated significant VR1/ER colocalization (70%).


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Fig. 4.   Only a subset of capsaicin- and thapsigargin-sensitive Ca2+ stores pharmacologically overlap in small sized DRG neurons. As shown in a, capsaicin releases Ca2+ from a Tg-insensitive store in small sized DRG neurons bathed in a nominally Ca2+-free medium. The trace of panel a is a population mean ± S.D. of the [Ca2+]i of seven cells from a single coverslip, which demonstrated Ca2+ mobilization in response to any of the indicated drug treatments and is representative of five separate experiments in different coverslips. As shown in b, considerable heterogeneity in the size of the capsaicin-sensitive/Tg-insensitive Ca2+ store was apparent in small sized DRG neurons analyzed (n = 94) with 34% of cells devoid of this store (left trace), 41.5% of cells possessing a smaller (middle trace) and 24.5% of cells a larger (right trace) capsaicin-sensitive/Tg-insensitive store than a Tg-sensitive Ca2+ store. Traces of panel b are responses of single small sized DRG neurons from the same coverslip and are representative of individual cell responses from five separate experiments. As shown in c, Tg releases Ca2+ from a capsaicin-insensitive store in small sized DRG neurons. The trace of panel c is a population mean ± S.D. of the [Ca2+]i of eight cells from a single coverslip, which demonstrated Ca2+ mobilization in response to any of the indicated drug treatments and is representative of three separate experiments in different coverslips. d, data derived from the experiments of panels a and c, showing proportions of small sized DRG neurons that possessed a capsaicin (Caps)-sensitive, a capsaicin-sensitive/Tg-insensitive, a Tg-sensitive, or a Tg-sensitive/capsaicin-insensitive Ca2+ store. e, Data derived from the experiments of panels a and c, showing the magnitude of the Ca2+ release from the same stores as in panel d, expressed as the mean maximum change in [Ca2+]i (Delta [Ca2+]i) ± S.E. In panels d and e, data for each bar were derived from at least three separate coverslip experiments, and numerical cell ratios and numbers of cells analyzed are indicated above each bar in panels d and e, respectively. *, p < 0.01 versus caps-sensitive store value, **, p < 0.001 versus Tg-sensitive store value (Student's t test for unpaired observations).

From the experiments of Fig. 4b, considerable heterogeneity was noted in the size of intrinsic Tg-sensitive and capsaicin-sensitive/Tg-insensitive Ca2+ stores in small sized cells (Fig. 4b). Of the 65% of cells that possessed a capsaicin-sensitive/Tg-insensitive store, this store was apparently larger than an intrinsic Tg-sensitive Ca2+ store in 37% of cells (23 out of 62) and smaller in the remainder. For cells that possessed both capsaicin- and Tg-sensitive Ca2+ stores, the main part of these stores were Tg- and capsaicin-insensitive (Fig. 4e), respectively. Although all small sized DRG neurons (n = 128) possessed a capsaicin-insensitive/Tg-insensitive/ionomycin-sensitive Ca2+ store, the amount of Ca2+ associated with this store appeared to be small (Fig. 4, a and c) as compared with the total ionomycin-releasable Ca2+ pool (Fig. 2b). Delta [Ca2+]i associated with release from the former (25 ± 16 (128) nM; mean ± S.D. (n)) was only 9% of that associated with release from the latter (277 ± 92 (38) nM; mean ± S.D. (n)), indicating that nearly all sequestered intracellular Ca2+ was mobilized by a sequential treatment of cells with 1 µM Tg followed by 1 µM capsaicin (or vice versa).

Although certain members of the TRP channel superfamily have been shown to be activated by Ca2+ store depletion, it remains controversial whether VR1 has an innate capacity to function as a SOCC. To determine whether VR1 might be regulated by the state of filling of intracellular Ca2+ stores, the effect of the VR1 antagonists ruthenium red (RR) and capsazepine (CPZ) on store depletion-activated Ca2+ influx and the effect of CPZ on store depletion-activated inward currents were assessed in small sized DRG neurons. Fura-2-loaded cells were transferred to a nominally Ca2+-free extracellular medium and were challenged with a bolus of Tg (1 µM) to deplete Ca2+ stores and activate store-operated Ca2+ influx (15-17). The latter was assessed by measuring the maximum change in [Ca2+]i (Delta [Ca2+]i) on introducing 1.8 mM Ca2+ back into the static incubation bath in untreated control cells and cells pretreated with either 10 µM RR (Fig. 5, a-c) or 10 µM CPZ (Fig. 5, d and e). In these experiments, we found that a new steady-state level of [Ca2+]i was attained after ~1000 s in the presence of 1 µM Tg, which was the minimal concentration required for maximal Ca2+ release from the dose-response curve of Fig. 1b. In RR-treated cells, Delta [Ca2+]i associated with Ca2+ introduction was significantly reduced, and cytosolic Ca2+ oscillations were observed in all cells analyzed (n = 35) as compared with untreated control cells (n = 39), which displayed no oscillation in [Ca2+]i (Fig. 5, a-c). A similar reduction in Delta [Ca2+]i was observed in CPZ-treated DRG neurons as compared with untreated controls (Fig. 5, d and e), although unlike RR-treated cells, cytosolic Ca2+ oscillations were not apparent in any CPZ-treated cell (n = 67) following the introduction of Ca2+.


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Fig. 5.   VR1 antagonists attenuate Ca2+ influx in Ca2+ store-depleted DRG neurons. In all experiments, cells were bathed in a nominally Ca2+-free medium in the presence of 1 µM Tg to deplete intracellular Ca2+ stores. As shown in a, treatment of store-depleted DRG neurons at 1000 s with the VR1 antagonist RR (10 µM) resulted in a smaller increase in [Ca2+]i following the introduction of 1.8 mM Ca2+ to the bath (right trace), as compared with untreated control cells (left trace). In b, the [Ca2+]i of at least 10 small sized DRG neurons was averaged for coverslip experiments of panel a to give individual population responses with each trace in panel b representing the mean ± S.D. of three population responses for untreated control (trace 1) and ruthenium red-treated (trace 2) cells. c, data derived from the experiments of panel a showing the mean maximum Delta [Ca2+]i ± S.E. following the introduction of 1.8 mM Ca2+ to the bath for 39 untreated control (white bar) and 35 ruthenium red-treated (black bar), small sized DRG neurons (*, t = -2.94, p = 0.004 versus control value; Student's t test for unpaired observations). d, as for panel b, except capsazepine was substituted for RR in experiments. e, data derived from the experiments of panel d showing the mean maximum Delta [Ca2+]i ± S.E. following the introduction of 1.8 mM Ca2+ to the bath for 52 untreated control (white bar) and 67 capsazepine-treated (black bar), small sized DRG neurons (*, t = -3.71, p = 0.0003 versus control value; Student's t test for unpaired observations).

The effect of CPZ on store depletion-activated inward currents induced by Tg was also assessed in capsaicin-sensitive small sized DRG neurons in the presence of extracellular Ca2+ (10 mM). From previous work, it was found that an extracellular Ca2+ concentration of 10 mM was required for a maximal store depletion-mediated inward current in rat basophilic leukemia cells (18). We therefore decided to use this concentration of Ca2+ in our bathing medium to permit the easy detection of store depletion-mediated inward currents in subsequent electrophysiology experiments. No detectable currents were elicited in the absence of Tg (1 µM) when intracellular [Ca2+]i was buffered by 5 mM EGTA in the patch pipette. Three types of current traces were observed in the 58 cells analyzed in the presence of Tg (Fig. 6, a-c). A small, sustained inward current that was maintained throughout the period of Tg exposure was observed in 17 out of 58 cells (Fig. 6a). About the same number of neurons (31%) failed to produce a detectable current in the presence of Tg (Fig. 6c). In contrast, a larger outward current preceded by a small inward current was recorded in 23 out of the 58 cells (Fig. 6b). Outward currents were abolished when Cs+ was substituted for K+ in the patch pipette. Therefore, the large Tg-induced outward conductance might be mediated by Ca2+-activated K+ channels following an increase in [Ca2+]i. To abolish the outward current induced by Tg, we replaced K+ with Cs+ in the patch pipette. Under these conditions, CPZ (10 µM) significantly reduced the Tg-induced inward current in all DRG cells tested (Fig. 6, d and e), in line with its effect on store depletion-activated Ca2+ influx in these cells (Fig. 5, d and e). In agreement with a previous report (1), we found that activation of a store depletion-mediated inward current by Tg did not affect subsequent capsaicin-induced currents in small sized DRG neurons. The mean amplitude of transient capsaicin-evoked responses in untreated control neurons was not significantly different from those in cells pretreated with Tg (1 µM) for 1000 s (4.3 ± 0.7 (13) nA versus 4.6 ± 0.9 (11) nA; mean ± S.D. (n); p > 0.1, Student's t test for unpaired observations).


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Fig. 6.   Inhibition of a Tg-induced inward current by capsazepine in capsaicin-sensitive small sized DRG neurons. Inward currents were detected by whole-cell patch clamp in the presence of extracellular Ca2+ (10 mM) with cells voltage-clamped at -70 mV. Only cells that elicited a transient inward current in response to 1 µM capsaicin at the end of the experiments were analyzed. Three types of current trace were observed in the presence of 1 µM Tg (58 cells analyzed). a, representative trace of a small, sustained inward current that was maintained throughout the period of Tg exposure (observed in 17 cells). b, representative trace of a larger outward current preceded by a small inward current (observed in 23 cells). As shown in c, no detectable current was observed in 18 cells, with a representative trace shown in panel c. As shown in d, to abolish any Tg-induced outward current, K+ was replaced with Cs+ in the patch pipette. Under these conditions, CPZ (10 µM) significantly reduced the Tg-induced inward current (p < 0.05, Student's t test for paired observations) in all (n = 6) DRG cells tested. In e, data bars represent the mean final plateau current evoked by 1 µM Tg ± S.E. of 17 naive control and 6 CPZ-treated small sized DRG neurons subjected to the same protocol as in panel d (*, p < 0.05 versus control value, Student's t test for paired observations).

To further test the hypothesis that VR1 functions as a SOCC, store depletion-activated Ca2+ influx and inward currents were investigated in HEK-293 cells transfected with green fluorescent protein (GFP) or cotransfected with GFP and VR1 (GFP/VR1). In all Ca2+ imaging experiments, cells were classed as VR1-expressing if they elicited an increase in [Ca2+]i to 1 µM capsaicin at the end of the experiment and were classed as GFP-expressing if they were fluorescent at the appropriate excitation wavelength for GFP (Ex = 490 nm, Em = 515 nm). In cotransfected cultures, 1 µM capsaicin induced an increase in [Ca2+]i or transient inward currents in all GFP-expressing cells (254 cells analyzed), thereby allowing us to use green fluorescence as a marker for functional VR1 expression. In the presence of extracellular Ca2+ (1.8 mM), there was no difference in basal [Ca2+]i levels between GFP/VR1-cotransfected/coexpressing cells and GFP-transfected/expressing control cells (205 ± 41 (85) nM versus 212 ± 43 (92) nM; mean ± S.D. (n); p > 0.5, Student's t test for unpaired observations). Furthermore, there was no difference in the basal manganese quench of cytosolic fura-2 fluorescence between both cell groups (data not shown), indicating no difference in basal Ca2+ influx and suggesting that VR1 was unlikely to be constitutively active in GFP/VR1-coexpressing cells. As for DRG neurons (see above), fura-2-loaded HEK-293 cells were transferred to a nominally Ca2+-free extracellular medium and were challenged with a bolus of Tg (1 µM) to deplete Ca2+ stores and activate store-operated Ca2+ influx. The latter was assessed by measuring the maximum change in [Ca2+]i (Delta [Ca2+]i) on introducing 1.8 mM Ca2+ back into the static incubation bath in GFP/VR1-cotransfected/coexpressing cells and GFP-transfected/expressing control cells (Fig. 7, a and c). Delta [Ca2+]i associated with Ca2+ introduction was significantly greater in GFP/VR1-cotransfected/coexpressing cells (n = 127), as compared with GFP-transfected/expressing control cells (n = 143) (Fig. 7, a and b). This enhanced store depletion-activated Ca2+ influx was abolished by pretreating GFP/VR1-coexpressing cells with 10 µM CPZ prior to the introduction of Ca2+ (Fig. 7, c and d), and capsaicin-induced increases in [Ca2+]i were substantially reduced at the end of experiments (Fig. 7c), consistent with CPZ antagonising VR1.


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Fig. 7.   VR1 expression enhances Ca2+ influx in Ca2+ store-depleted HEK-293 cells. In all experiments, cells were bathed in a nominally Ca2+-free medium in the presence of 1 µM Tg to deplete intracellular Ca2+ stores. As shown in a, enhanced Ca2+ influx was observed in store-depleted GFP/VR1-cotransfected/coexpressing cells as compared with control GFP-transfected/expressing cells following the introduction of 1.8 mM Ca2+ to the bathing medium. [Ca2+]i of at least 40 cells was averaged for coverslip experiments to give individual population responses with each trace in panel a representing the mean ± S.D. of three population responses for control GFP-transfected/expressing cells (trace 1) and GFP/VR1-cotransfected/coexpressing cells (trace 2). b, data derived from the experiments of panel a showing the mean maximum Delta [Ca2+]i ± S.E. following the introduction of 1.8 mM Ca2+ to the bath for 143 control GFP-transfected/expressing cells (white bar) and 127 GFP/VR1-cotransfected/coexpressing cells (black bar) (*, t = 11.95, p < 0.0001 versus GFP-transfected value, Student's t test for unpaired observations). c, as for panel a, except 10 µM CPZ was added to GFP-transfected/expressing cells (trace 1) and GFP/VR1-cotransfected/coexpressing cells (trace 2) at the indicated time prior to the introduction of Ca2+ to the bath. d, data derived from the experiments of panel c showing the mean maximum Delta [Ca2+]i ± S.E. on introducing 1.8 mM Ca2+ to the bath for 98 control GFP-transfected/expressing cells (white bar) and 103 GFP/VR1-cotransfected/coexpressing cells (black bar) in the presence of 10 µM CPZ (*, t = 1.24, p = 0.22 versus GFP-transfected + 10 µM CPZ value, Student's t test for unpaired observations). e, fura-2 ratio image sequence of a GFP/VR1-cotransfected culture subjected to the experimental protocol indicated in the corresponding bottom traces. The bottom traces display the mean [Ca2+]i ± S.D. of 47 capsaicin-insensitive (trace 1) and 45 capsaicin-sensitive (trace 2) cells randomly selected from the field of view of the ratio image sequence. The bar graph in the inset represents data derived from the imaging experiment and shows the mean maximum Delta [Ca2+]i ± S.E. following the introduction of 1.8 mM Ca2+ to the bath for the same 47 capsaicin-insensitive (white bar) and 45 capsaicin-sensitive (black bar) HEK-293 cells randomly selected from the field of view (*, t = 11.95, p < 0.0001 versus capsaicin-insensitive value, Student's t test for unpaired observations). Data in panel e are representative of three separate coverslip experiments for GFP/VR1-cotransfected cultures. Scale bar = 50 µm.

In GFP/VR1-cotransfected cultures, ~40% of cells elicited an increase in [Ca2+]i to 1 µM capsaicin at the end of experiments (Fig. 7e), indicating that the remaining unresponsive cells did not functionally express VR1. Comparing Delta [Ca2+]i associated with Ca2+ introduction in capsaicin-sensitive and -insensitive cells from the same coverslip revealed greater Ca2+ influx for cells functionally expressing VR1 (Fig. 7e) as compared with cells that were capsaicin-insensitive, consistent with the above data derived from comparing GFP-transfected and GFP/VR1-cotransfected cultures (Fig. 7, a and b).

From whole-cell voltage clamp experiments and in contrast to DRG neurons, GFP-transfected/expressing control HEK-293 cells did not respond to 1 µM Tg with a detectable inward current (Fig. 8a) in any cell analyzed (n = 24). This might explain a 47% lower store depletion-mediated Delta [Ca2+]i for control HEK-293 cells (Fig. 7b) as compared with DRG neurons (395 ± 13 (143) nM versus 843 ± 50 (39) nM; mean ± S.E. (n); p < 0.0001, Student's t test for unpaired observations) (Fig. 5c) in Ca2+ introduction experiments. As expected, GFP-transfected/expressing control cells were unresponsive to 1 µM capsaicin with this treatment failing to evoke an inward current (Fig. 8a) in any cell analyzed (n = 24). In contrast to the above, 13 out of 24 GFP/VR1-coexpressing cells elicited a sustained inward current in response to 1 µM Tg (Fig. 8b). As for DRG neurons, Tg-induced currents slowly reached a maximum sustained plateau (39 ± 7 (13) pA; mean ± S.E. (n); 10-80 pA current range), and capsazepine treatment (10 µM) completely abrogated this plateau current (Fig. 8b) in all cells analyzed (n = 13), in line with its effect on store depletion-activated Ca2+ influx in these cells (Fig. 7, c and d).


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Fig. 8.   Inhibition of a Tg-induced inward current by capsazepine in VR1-expressing HEK-293 cells. Inward currents were detected by whole-cell patch clamp in the presence of extracellular Ca2+ (10 mM) with cells voltage-clamped at -70 mV. For GFP/VR1-cotransfected cultures, cells were classed as VR1-expressing if they elicited a transient inward current to 1 µM capsaicin (Caps) at the beginning of the experiment (shown in the right-hand trace of panel b). As shown in a, all GFP-transfected control cells (solely expressing GFP) were devoid of a Tg-induced current (24 out of 24 cells analyzed) with a representative single cell trace shown. As shown in b, Tg (1 µM) induced a sustained inward current in most GFP/VR1-coexpressing HEK-293 cells (13 out of 24 cells analyzed). This sustained Tg-induced inward current was completely abrogated by the addition of 10 µM capsazepine to the incubation bath, with the trace in panel b representative of current abrogation observed in 13 out of 13 different cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Combined data from this study are supportive of a multifaceted role for VR1 in regulating [Ca2+]i in small sized DRG neurons with topographical cellular location possibly dictating whether this receptor functions as a facilitator of Ca2+ release from intracellular stores or Ca2+ influx across the plasma membrane. In agreement with a previous study (7), the VR1 receptor agonist capsaicin induced Ca2+ mobilization from intracellular stores of small sized rat DRG neurons in the absence of extracellular Ca2+. However, in contrast to this study, the EC50 for capsaicin-induced Ca2+ release estimated by Eun et al. (7) was considerably higher than that estimated here (13.5 ± 0.22 µM versus 133.1 ± 20.4 nM). This apparent discrepancy might be due to differences in experimental protocol and the data acquired in the two studies. In this study, we used a static incubation protocol employing single bolus additions of capsaicin to different coverslip cultures, whereas in the study of Eun et al. (7), single cells were subjected to multiple capsaicin additions of varying concentrations using a perfused system. The latter protocol may have resulted in VR1 desensitization as it is well established that sensory neurons are desensitized by capsaicin (19, 20) via a Ca2+-dependent process (21). With repeated applications of capsaicin, subsequent increases in [Ca2+]i of DRG neurons show marked attenuation, and this becomes more pronounced at higher capsaicin concentrations (22). It is therefore possible that VR1 desensitization might have resulted in an overestimation of the EC50 vlaue for capsaicin-induced Ca2+ release in the dose-response study of Eun et al. (7). It is noteworthy that the value of this parameter estimated in our study is similar to the EC50 values for capsaicin-induced increases in [Ca2+]i in the presence of extracellular Ca2+ (22), plasma membrane depolarization (23), and inward currents (23), indicating the likelihood that all the above dose responses were due to the activation of the same or a similar receptor-operated conductance. Additional data were also consistent with VR1-capsaicin binding inducing Ca2+ release in small sized DRG neurons: capsaicin-induced Ca2+ release was confined to DRG neurons in the cultures of this study and was not evident in glial cells, suggesting that it was sensory neuron-specific, and the response was abrogated by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca2+ mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation.

From confocal microscopy experiments, extensive colocalization for VR1 and ER was detected in the majority of small sized DRG neurons analyzed (Fig. 3), suggesting that an increase in the Ca2+ permeability of Ca2+ stores might be facilitated by the direct binding of capsaicin to store-associated VR1, resulting in an activated conducting state for this form of the receptor. These data do not, however, preclude the possibility that capsaicin-mediated Ca2+ release proceeds via plasma membrane-associated VR1 binding as this might induce Ca2+ mobilization by an as yet unidentified signaling mechanism involving communication between the plasma membrane and intracellular Ca2+ stores. It is notable, however, that a similar proportion of small sized DRG neurons displayed colocalization for ER and VR1 (70%) as that which possessed capsaicin-sensitive intracellular Ca2+ stores (74%), perhaps implying a correlation between capsaicin-induced Ca2+ release and ER/VR1 association. These combined data suggest that ER-associated VR1 might function as a Ca2+ release receptor in sensory neurons. This hypothesis is both timely and pertinent in view of the recent identification of the endogenous capsaicin-like VR1 agonist, N-arachidonyl-dopamine, which is biosynthesised in mammalian nervous tissues and is present at a low basal concentration in dorsal root ganglia (24). It remains to be established, however, whether N-arachidonyl-dopamine might function as an intracellular Ca2+ mobilizing second messenger, capable of releasing Ca2+ from VR1-linked Ca2+ stores in DRG neurons.

Further analysis of Ca2+ mobilization in small sized DRG neurons revealed pharmacological heterogeneity for the Ca2+ stores of these cells. Although a subset of capsaicin- and Tg-sensitive intracellular Ca2+ stores demonstrated pharmacological overlap (inferred from Fig. 4e), the main part of these stores were Tg- and capsaicin-insensitive, respectively, suggesting that ER-associated VR1 might be predominantly localized to a Tg-insensitive Ca2+ pool in these cells. Our data were also consistent with Tg or capsaicin sensitivity, accounting for the majority of the Ca2+ stores of a typical small sized DRG neuron as sequential additions of these agents mobilized the main part of the total releasable Ca2+ pool of these cells (Fig. 4). Previous studies have also demonstrated Tg insensitivity for Ca2+ stores in other mammalian cell types (25-27). Endogenous non-mitochondrial Ca2+ stores of A7r5 and 16HBE14o cells have been shown to be Tg-insensitive, consistent with the demonstrated expression of a mammalian homologue of the Tg-insensitive Golgi Ca2+ pump Pmr1, in both cell types (26). It is therefore conceivable that the capsaicin-sensitive/Tg-insensitive Ca2+ store of small sized DRG neurons may be charged by a Ca2+ pump homologous to Pmr1, and in view of the proposed ryanodine sensitivity for capsaicin-induced Ca2+ release (7), it might share similar membrane composition to the ryanodine-sensitive and Tg-insensitive sarcoplasmic reticulum Ca2+ store identified in cardiac myocytes (27).

In small sized DRG neurons that displayed a capsaicin-sensitive/Tg-insensitive Ca2+ store, considerable heterogeneity was noted in the size of this store and of the intrinsic Tg-sensitive Ca2+ store (Fig. 4b) with the size of the total releasable Ca2+ pool of cells remaining invariant (inferred from Fig. 4e). This might be a reflection of differential functional expression for VR1 within ER membranes with greater expression somehow resulting in a reduction in the size of the Tg-sensitive Ca2+ pool. In this way, the size of the total releasable Ca2+ pool of DRG neurons would be maintained within close limits, which is consistent with the data from this study (Fig. 4e). Although the precise functional implications of such store heterogeneity in small sized cells are not apparent from this study, it could be argued that cells with ER-associated VR1 may be more susceptible to chemical (vanilloids and pH) and heat-induced desensitization and/or cell death, due perhaps to Ca2+ release from VR1-linked Ca2+ stores contributing to a larger and more sustained increase in [Ca2+]i. Additional work beyond the scope of this study would be required to test this hypothesis.

From the analysis of inward currents and Ca2+ influx in Ca2+ store-depleted DRG neurons, combined data suggest that the activity of VR1 is linked to the state of filling of intracellular Ca2+ stores. In DRG neurons, both CPZ and RR significantly decreased Ca2+ influx associated with Ca2+ store depletion in Ca2+ introduction experiments, and CPZ attenuated a sustained Tg-induced inward current, suggesting an involvement for VR1 in store depletion-activated Ca2+ influx in these cells. It is notable, however, that VR1 antagonists failed to completely abrogate the above responses, indicating the likely involvement of additional cation conductances in these cells. In contrast to CPZ, treatment of store-depleted cells with RR resulted in intracellular Ca2+ oscillations following bath introduction of Ca2+. Although the reason for this is not obvious, it is well accepted that RR also inhibits Ca2+ release from ryanodine receptor-linked intracellular stores (28), and this type of store may not have been functionally eliminated by the prior treatment of cells with Tg, due to the presence of Tg-insensitive Ca2+ pumps (see above). If RR binding and subsequent inhibition of plasma membrane/ER-associated VR1 or ryanodine receptors is dynamically controlled by [Ca2+]i in these cells, then this might account for the observed Ca2+ oscillations in RR-treated cells.

In addition to VR1, it is becoming increasingly apparent that DRG neurons possess a variety of other TRP channel subtypes, including temperature-sensitive vanilloid receptor like protein 1 and 3 (VRL-1, VRL-3) (29-31) and the cold- and menthol-sensitive receptor (CMR1) (32). It is possible that these non-selective cation channels may also contribute to store depletion-activated Ca2+ influx in DRG neurons. It should be noted that a major group of SOCCs, termed Ca2+ release-activated Ca2+ channels, are thought to be among the most selective for Ca2+ (33). However, from electrophysiological studies, there are other SOCCs that have properties distinct from those of Ca2+ release-activated Ca2+ channels (34). Furthermore, CaT1 (or TRPV6) is a member of the same structural group of TRP channels (TRPV) as VR1, VRL-1, and VRL-3 and has been proposed to be regulated by Ca2+ store depletion (35). It is clearly evident that only a vague molecular definition of SOCCs exists at this point in time.

Further compelling evidence that VR1 is regulated by the state of filling of intracellular Ca2+ stores was derived from Ca2+ imaging and electrophysiological experiments using VR1-transfected HEK-293 cells. When compared with GFP-expressing control cells, greater Ca2+ influx was observed in GFP/VR1-coexpressing cells following Ca2+ store depletion, and this was attenuated by prior treatment of cells with CPZ (Fig. 7). Furthermore, Tg failed to induce inward currents in GFP-expressing control cells, whereas Tg-induced inward currents of GFP/VR1-coexpressing cells were abrogated by CPZ (Fig. 8). These data are in direct conflict with a previous study that failed to detect activation of expressed VR1 following Tg-induced Ca2+ store depletion in Xenopus oocytes (1). Although the precise reason for these differing observations is not clear, we can only speculate that the regulation of VR1 might be cell-specific, with there perhaps being little or no communication between intracellular Ca2+ stores and plasma membrane-associated VR1 in Xenopus oocytes. Data from this study suggest that store-operated and capsaicin-induced activation of VR1 may proceed via a similar mechanism as CPZ was effective in attenuating store depletion-activated Ca2+ influx and inward currents in DRG neurons and VR1-transfected HEK cells. For plasma membrane-associated VR1, it is likely that the binding site for capsaicin is on the intracellular side of the membrane (36), and activation has been proposed to involve the sixth transmembrane pore domain, which transduces capsaicin binding to channel gating (37). It remains to be established, however, whether capsaicin and store-operated activation of VR1 share a similar mechanistic profile, although further study in this area may shed light on how store depletion results in SOCC activation in general.

In conclusion, this study highlights topographical and functional diversity for VR1 in the regulation of [Ca2+]i of small sized DRG neurons. Combined data suggest that activation of ER-associated VR1 by capsaicin binding results in Ca2+ mobilization from an intracellular Ca2+ store, which we tentatively assign as predominantly Tg-insensitive. This raises the possibility that VR1 may function as a Ca2+ release receptor in small sized DRG neurons. Additionally, this study presents compelling evidence that plasma membrane-associated VR1 is regulated by the state of filling of intracellular Ca2+ stores. We therefore propose that this form of the receptor functions as a SOCC in small sized DRG neurons.

    FOOTNOTES

* This work was supported by grants from the Wellcome Trust and the Royal London Hospital Special Trustees.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-20-7882-6991; Fax: 44-20-7982-7726; E-mail: n.j.willmott@qmul.ac.uk.

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M209111200

    ABBREVIATIONS

The abbreviations used are: VR1, vanilloid receptor 1; VRL-1, vanilloid receptor-like protein 1; VRL-3, vanilloid receptor-like protein 3; CMR1, cold- and menthol-sensitive receptor 1; DRG, dorsal root ganglion; ER, endoplasmic reticulum; HEK-293, human embryonic kidney-293; TRP, transient receptor potential; TRPV, TRP, channel type V; SOCC, store-operated Ca2+ channel; RyR, ryanodine receptor; [Ca2+]i, free cytosolic Ca2+ concentration; Delta [Ca2+]i, maximum change in the free cytosolic Ca2+ concentration; GFP, green fluorescent protein; EGFP, enhanced GFP; pEGFP, GFP plasmid; PBS, phosphate buffered saline; HBSS, Hanks' buffered salt solution; pcDNA3.1/rVR1, VR1 plasmid; DiOC5, 3,3'-dihexyloxacarbocyanine iodide; TRITC tetramethyl rhodamine isothiocyanate, IgG, immunoglobulin G; Tg, thapsigargin; CPZ, capsazepine; RR, ruthenium red.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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