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Originally published In Press as doi:10.1074/jbc.M501229200 on March 3, 2005

J. Biol. Chem., Vol. 280, Issue 18, 18142-18151, May 6, 2005
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Transient Receptor Potential Vanilloid Type 1 Activation Down-regulates Voltage-gated Calcium Channels through Calcium-dependent Calcineurin in Sensory Neurons*

Zi-Zhen Wu, Shao-Rui Chen, and Hui-Lin Pan, Recipient of a National Institutes of Health Independent Scientist Career Award{ddagger}

From the Department of Anesthesiology, Department of Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Received for publication, February 2, 2005 , and in revised form, March 2, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium influx through voltage-activated Ca2+ channels (VACCs) plays a critical role in neurotransmission. Capsaicin application inhibits VACCs and desensitizes nociceptors. In this study, we determined the signaling mechanisms of the inhibitory effect of capsaicin on VACCs in primary sensory neurons. Whole-cell voltage clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Capsaicin caused a profound decrease in the Ca2+ current (ICa) density in capsaicin-sensitive, but not -insensitive, dorsal root ganglion neurons. At 1 µM, capsaicin suppressed about 60% of N-, P/Q-, L-, and R-type ICa density. Pretreatment with iodoresiniferatoxin, a specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked the inhibitory effect of capsaicin on Ica. However, neither W-7, a calmodulin blocker, nor KN-93, a CaMKII inhibitor, attenuated the inhibitory effect of capsaicin on ICa. Furthermore, intracellular dialysis of deltamethrin or cyclosporin A, the specific calcineurin (protein phosphatase 2B) inhibitors, but not okadaic acid (a selective protein phosphatase 1/protein phosphatase 2A inhibitor), abolished the effect of capsaicin on ICa. Interestingly, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, deltamethrin, cyclosporin A, and okadaic acid each alone significantly increased the ICa density and caused a depolarizing shift in the voltage dependence of activation. Immunofluorescence labeling revealed that capsaicin induced a rapid internalization of CaV2.2 channels on the membrane. Thus, this study provides novel information that VACCs are tonically modulated by the intracellular Ca2+ level and endogenous phosphatases in sensory neurons. Stimulation of TRPV1 by capsaicin down-regulates VACCs by dephosphorylation through Ca2+-dependent activation of calcineurin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRPV1 1 is a nonselective cation channel with high Ca2+ permeability and is the molecular target of capsaicin, the main pungent ingredient in chili peppers. The TRPV1 channel is expressed in subsets of primary sensory neurons and nerve terminals and plays an essential role in detecting noxious heat and several other nociceptive stimuli (1, 2). Capsaicin can excite nociceptive sensory neurons and produce transient pain in animals and humans (3, 4). Paradoxically, exposure to capsaicin desensitizes nociceptive sensory neurons and results in long lasting pain relief. For example, topical or local application of capsaicin is effective in treating many acute and chronic pain syndromes in patients (5–7). Capsaicin also causes an unexplained synaptic transmission block in the spinal cord dorsal horn (8).

The voltage-activated Ca2+ channels (VACCs) play a critical role in signal transduction, synaptic neurotransmitter release, and nociceptive transmission (9–11). VACCs also are an important molecular target of many analgesic drugs such as opioids (12, 13). Interestingly, capsaicin causes a profound inhibition of VACC currents in dorsal root ganglion (DRG) neurons (14). However, the cellular and signaling mechanisms of the capsaicin effect on VACCs remain poorly understood.

Protein kinases and phosphatases are key enzymes in signal transduction pathways for a wide range of cellular processes. The enzymatic addition or removal of phosphate esters on serine and threonine hydroxyls alters the activity of many proteins that are essential to the characteristic structure and function of neurons. An important mechanism regulating VACC function is through phosphorylation by protein kinases and phosphatases (15–18). We now show that a Ca2+-dependent serine/threonine phosphatase, calcineurin (protein phosphatase 2B), is critically involved in down-regulation of high voltage-activated Ca2+ channels (HVACCs) by capsaicin in native DRG neurons. Furthermore, the basal intracellular Ca2+ level and endogenous protein phosphatases tonically modulate the HVACC current in DRG neurons. These findings are not only important to our understanding of the functional interaction between TRPV1 and HVACCs in primary nociceptors but are also significant to our understanding of the Ca2+-dependent feedback regulation of neuronal Ca2+ channels in general.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of DRG Neurons—All procedures conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine. Male Sprague-Dawley rats (5–6 weeks old; Harlan, Indianapolis, IN) were anesthetized with halothane and then rapidly decapitated. The thoracic and lumbar segments of the vertebrate column were dissected. The DRGs were quickly removed and transferred immediately into Dulbecco's modified Eagle's medium (Invitrogen). After removal of attached nerves and surrounding connective tissues, the DRGs were minced with fine spring scissors, and the ganglion fragments were placed in a flask containing 5 ml of Dulbecco's modified Eagle's medium in which trypsin (type I, 0.5 mg/ml; Sigma), collagenase (type IA, 1 mg/ml; Sigma), and DNase (type I, 0.1 mg/ml; Sigma) had been dissolved. After incubation at 34 °C in a shaking water bath for 30 min, soybean trypsin inhibitor (type II-s, 1.25 mg/ml; Sigma) was then added to stop trypsin digestion. The cell suspension was centrifuged (500 rpm, 5 min) to remove the supernatant and replenished with Dulbecco's modified Eagle's medium. The cultured DRG cells were replenished with B-27 medium containing neurobasal medium (Invitrogen) with B-27 supplement (2%; Invitrogen) and penicillin/streptomycin/glutamine supplement (1%; Invitrogen). Cells were then plated onto a 35-mm culture dish containing poly-L-lysine (50 µg/ml) precoated coverslips and kept for at least 30 min before electrophysiological recordings. Recordings were made within 24 h after dissociation to keep the experiment as similar to in vivo as possible and to minimize the space clamp error, since DRG neurons produce neurites that are difficult to clamp following prolonged neuronal culture.

Electrophysiological Recordings—Patch electrodes with a resistance of ~2 megaohms were pulled from GC150TF-10 glass capillaries (inner diameter 1.17 mm, outer diameter 1.5 mm; Harvard Apparatus, Holliston, MA) using a micropipette puller (P-97 Sutter Instrument Co., Novato, CA) and fire-polished (DMF1000; World Precision Instruments, Sarasota, FL). Immediately before recording, neurons were treated with IB4-Alexa 594 (3 µg/ml; Molecular Probes, Inc., Eugene, OR) in Tyrode solution for 10 min and then rinsed for at least 3 min. IB4-positive neurons were identified using a combination of epifluorescence illumination and differential interference contrast (x20–40) optics on an inverted IX70 microscope (Olympus Optical, Tokyo, Japan). Images of cells were taken with a CCD camera and displayed on a video monitor. Neurons were recorded in the whole-cell configuration using an EPC-10 amplifier (HEKA Instruments). After whole-cell configuration was established, the cell membrane capacitance and series resistance were electronically compensated. All experiments were performed at room temperature (~25 °C). Leak currents were subtracted using a hyperpolarizing P/4 protocol. Signals were filtered at 1 kHz, digitized at 10 kHz, and acquired using the Pulse program (HEKA).

Calcium currents (ICa) were recorded using extracellular solution consisting of 140 mM tetraethylammonium (TEA)-Cl, 2 mM MgCl2, 5 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH 7.4 adjusted with TEA-OH, osmolarity 320 mosM). In some cells, barium was used as the charge carrier to record Ba2+ currents (IBa) flowing through Ca2+ channels. The extracellular solution consisted of 140 mM TEA, 2 mM MgCl2, 3 mM BaCl2, 10 mM glucose, 10 mM HEPES (pH 7.4 adjusted with TEA-OH, osmolarity 320 mosM). The pipette internal solution contained 120 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 10 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na-GTP (pH 7.2 adjusted with CsOH, osmolarity 300 mosM). GTP and ATP were included in the pipette solution to minimize the "run-down" associated with the whole-cell recording. The whole-cell Ca2+ current, carried by calcium (ICa) or barium (IBa), was elicited by a series of command potentials from -70 to 50 mV for 150 ms in 10-mV steps (5-s intervals) from a holding potential of -90 mV. The steady-state inactivation of VACCs was obtained by depolarizing cells to a series of prepulse potentials from -100 to 20 mV for 500 ms followed by a command potential to 0 for 150 ms.

Most drugs were dissolved in distilled water at 1,000 times the final concentration and kept frozen in aliquots. Nimodipine, thapsigargin, and KN-93 were initially dissolved in Me2SO to make the stock solution. The capsaicin, cyclosporine A, and deltamethrine stock solutions were prepared using ethanol. The stock solutions were further diluted in the extracellular or intracellular solution just before use and held in a series of independent syringes connected to corresponding fused silica columns. The end of the parallel columns was connected to a common silica column. The distance from the column mouth to the cell examined was about 100 µm. Cells in the recording chamber were continuously bathed in extracelluar solution. Each drug solution was delivered to the recording chamber by gravity, and rapid solution exchange was achieved by controlling the corresponding valve switch (World Precision Instruments). Drugs and chemicals were purchased from Sigma, except {omega}-conotoxin GVIA, {omega}-agatoxin IVA, and {omega}-conotoxin MVIIC (Alomone Labs, Jerusalem, Israel) and W-7 and deltamethrine (Calbiochem).

Double Fluorescence Labeling of CaV2.2 and TRPV1/IB4 in DRG Cells—The N-type current (CaV2.2 channel) is the most predominant subtype of VACC currents in DRG neurons (22). We determined the effect of capsaicin on the spatial distribution of CaV2.2 using immunofluorescence and confocal microscopy. DRG neurons cultured on glass coverslips were rinsed three times with phosphate-buffered saline. The cells were incubated with 1–10 µM capsaicin or vehicle in phosphate-buffered saline for 20 s or 2 min. Additional cells were pretreated with either 10 µM iodoresiniferatoxin, a specific TRPV1 antagonist (19), or 1 µM deltamethrin, a membrane-permeable inhibitor of calcineurin (20), for 5 min before capsaicin incubation. After rinsing, the cells were immediately fixed with 4% formaldehyde for 10 min. The cells were quenched in 1% H2O2 in Tris-buffered saline for 10 min and permeabilized in 0.1% Triton X-100 in Tris-buffered saline for another 10 min at room temperature. Then the cells were blocked in 4% normal goat serum for 30 min at room temperature.

For CaV2.2 and IB4 double labeling, the cells were incubated overnight at 4 °C with the primary antibody (rabbit anti-CaV2.2, 1:200 dilution; Chemicon (Temecula, CA) or Alomone (Jerusalem, Israel)) diluted in 2% normal goat serum. Subsequently, the cells were washed and incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:100 dilution; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The cells were then rinsed and incubated with fluorescein isothiocyanate-conjugated tyramide (1:100 dilution; PerkinElmer Life Sciences) for 5 min at room temperature and washed. Finally, the cells were incubated with Alexa 594 conjugated to IB4 (1 µg/ml; Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature.

For CaV2.2 and TRPV1 double immunolabeling, the cells were incubated overnight at 4 °C with a mixture of two primary antibodies (rabbit anti-CaV2.2 and guinea pig anti-TRPV1, 1:1000 dilution; Neuromics, Northfield, MN). The cells were first processed with the CaV2.2 staining as described above. The cells were then incubated with biotin-conjugated goat anti-guinea pig IgG (1:200 dilution; Jackson ImmunoResearch) for 1 h at room temperature. After rinsing, cells were incubated with Alexa 594 conjugated to streptavidin for 1 h at room temperature. Subsequently, the slides were covered and mounted with the ProLong mounting medium. Negative controls were performed by omitting the primary antibodies. The labeled cells were examined on a laser-scanning confocal microscope (Leica, Germany), and areas of interest were photodocumented. Confocal laser scanning microscopy was used for accurate localization of fluorescent markers, because the thin (0.3-µm) optical sectioning generated by the confocal microscope eliminates the confounding effect of out-of-focus fluorescence.

Data Analysis—Data were analyzed using the PulseFit software program (HEKA). Whole-cell current-voltage (I-V) relationships for individual neurons were constructed by calculating the mean peak inward current at each test potential and normalized for cell capacitance. Conductance-voltage (G-V) curves were calculated by dividing current at each potential by the driving force (V - Vr), where V is the test potential and Vr is the reversal potential extrapolated from the I-V curve. The normalized conductance (G/Gmax) for G-V relationships and inactivation curves were fitted with a Boltzmann function G/Gmax = Gmin + (1/(1 + exp(V - V0.5)/k))), where Gmin is the minimal conductance of VACCs, Gmax is the maximal conductance, V0.5 is the voltage for 50% activation or inactivation of VACCs, and k is a voltage-dependent slope factor. The percentage inhibition of total ICa and subtypes of ICa was calculated as the ratio of capsaicin-inhibited ICa to the total peak ICa or subtypes of VACC currents during control, respectively. Statistical data are presented as means ± S.E. All comparisons between means were tested for significance using Student's unpaired t test or one-way analysis of variance unless otherwise indicated. p < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Capsaicin Inhibits VACC Currents through TRPV1—The whole-cell VACC currents were elicited by a series of depolarizing pulses (from -70 to 50 mV for 150 ms in 10-mV increments) from a holding potential of -90 mV. The inward ICa was typically activated at -30 mV and reached its maximum at 0 mV (Fig. 1). TRPV1 is expressed predominantly in isolectin B4 (IB4)-binding DRG neurons (21). Because IB4 can label living rat DRG neurons (15–30 µm in diameter) and does not affect VACC currents (22, 23), we used IB4 as a marker to identify capsaicin-sensitive DRG neurons.

In the IB4-positive cells included for this study, 1 µM capsaicin produced inward currents ranging from 200 to 1,000 pA (406.3 ± 14.7 pA, n = 108). Consistent with the previous study (24), we did not observe the low voltage-activated (T-type) Ca2+ currents in capsaicin-sensitive small diameter DRG neurons. Thus, only HVACC currents were studied before capsaicin application and 1–2 min after washing of the capsaicin. The application of 1 µM capsaicin caused a large inhibition of ICa density in all 15 capsaicin-sensitive neurons tested (Fig. 1A). Capsaicin inhibited ICa density by 55.7 ± 6.9 and 60.9 ± 6.8% at 0 and 10 mV, respectively, in these capsaicin-sensitive neurons (Fig. 1B). The inhibitory effect of ICa by capsaicin was partially washed out ~3 min after capsaicin application. Prolonged exposure to capsaicin for more than 3 min led to complete inhibition of ICa, which did not recover after washing. Although capsaicin did not alter the I-V curve, it shifted the reversal potential from 50.5 ± 1.7 to 41.7 ± 3.1 mV (Fig. 1B). In contrast, in seven capsaicin-insensitive DRG neurons, 1 µM capsaicin had no significant effect on ICa (Fig. 1C). Furthermore, capsaicin caused a similar degree of ICa inhibition in nine IB4-negative but capsaicin-sensitive DRG neurons (Fig. 2C).



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  		FIG. 1.
Capsaicin inhibits HVACC currents in IB4-positive DRG neurons. A, representative traces showing ICa before and after application of 1 µM capsaicin in a capsaicin-sensitive DRG neuron. B, mean ICa density before and after application of capsaicin at different test potentials (n = 15). C, original recordings of ICa before and after capsaicin application in a capsaicin-insensitive neuron. D, treatment with 500 ng/ml pertussis toxin had no effect on capsaicin-induced inhibition of ICa (n = 9). The peak current was divided by cell capacitance to yield the current density. All of the neurons were voltage-clamped at -90 mV and depolarized from -70 to 50 mV for 150 ms with 10-mV increments. Inset, the double-pulse voltage protocol with a depolarizing prepulse to 100 mV for 50 ms preceding the second test pulse. *, p < 0.05 compared with corresponding values before capsaicin application.

 
Many G protein-coupled receptor agonists, including opioids, inhibit HVACC currents of DRG neurons (12, 22). It has been demonstrated that G protein {beta}{gamma}, rather than the G{alpha}, subunit is responsible for inhibition of VACCs by G proteins. With a high voltage prepulse, the G{beta}{gamma} subunit is believed to release from the Ca2+ channel {alpha}1 subunit, thereby relieving the tonic inhibition of VACCs by G proteins and producing "prepulse facilitation" (25, 26). We used a double-pulse voltage protocol (27) to determine whether G proteins are involved in the effect of capsaicin on ICa. The voltage dependence of capsaicin-induced inhibition of ICa was examined by a depolarizing prepulse to 100 mV, preceding the second test pulse (Fig. 1A). There was no voltage-dependent relief of inhibition on ICa by capsaicin when a strong conditioning depolarization (+100 mV) prepulse was used (Fig. 1A, n = 15). Using this protocol, 1 µM capsaicin produced a similar degree of inhibition of ICa (Fig. 1, A and B). Furthermore, in nine separate DRG neurons treated with pertussis toxin (500 ng/ml) overnight to inactivate inhibitory G proteins, 1 µM capsaicin still significantly inhibited ICa (57.9 ± 10.1% at 0 mV; Fig. 1D).

To determine whether the effect of capsaicin on ICa is specifically mediated by TRPV1, we used a highly specific TRPV1 antagonist, iodoresiniferatoxin (19). Iodoresiniferatoxin (10 µM) alone did not produce any currents and had no effect on ICa (Fig. 2, A and B). In the presence of 10 µM iodoresiniferatoxin, 1 µM capsaicin failed to produce any inward currents and had no inhibitory effect on ICa in all 13 IB4-positive neurons examined (Fig. 2, A and B).

Capsaicin Inhibits All Subtypes of HVACCs—We next determined which subtypes of HVACC currents are inhibited by capsaicin. The subtype-selective Ca2+ channel blockers nimodipine (5 µM, L-type), {omega}-conotoxin GVIA (2 µM, N-type), {omega}-agatoxin IVA (100 nM, P/Q-type), and {omega}-conotoxin MVIIC (500 nM, N- and P/Q-type) were appropriately combined to pharmacologically isolate L-, N-, P/Q-, and R-type Ca2+ currents, as described previously (22, 28, 29). Because 100 nM {omega}-agatoxin IVA alone is not sufficient to block the Q-type Ca2+ channel (29), {omega}-conotoxin MVIIC was co-applied with {omega}-agatoxin IVA to define L- and R-type VACC currents. In this protocol, the cells were depolarized from -90 to 0 mV for 150 ms. A desired subtype of ICa was isolated before application of 1 µM capsaicin for 30 s. After capsaicin current was washed out, the ICa was reexamined in capsaicin-sensitive neurons.

To determine the effect of 1 µM capsaicin on R-type ICa, {omega}-conotoxin MVIIC and {omega}-agatoxin IVA were co-applied with nimodipine and {omega}-conotoxin GVIA (Fig. 3). The R-type (drug-resistant) ICa was very small in DRG neurons, and 1 µM capsaicin suppressed 58.5 ± 12.6% R-type ICa (n = 6). Cd2+ (300 µM) completely blocked the remaining ICa. The N-type ICa was isolated by application of nimodipine and {omega}-agatoxin IVA in a separate group of DRG neurons. Capsaicin inhibited 61.9 ± 11.6% N-type ICa in nine DRG neurons tested (Fig. 3). Furthermore, the L-type Ca2+ current, isolated with {omega}-conotoxin GVIA, {omega}-conotoxin MVIIC, and {omega}-agatoxin IVA, was inhibited 48.5 ± 8.9% (n = 5; Fig. 3) by 1 µM capsaicin. Additionally, 1 µM capsaicin inhibited 57.1 ± 14.9% P/Q-type Ca2+ current, isolated using {omega}-conotoxin GVIA and nimodipine (n = 5; Fig. 3).



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  		FIG. 2.
Iodoresiniferatoxin (I-RTX) blocked the inhibitory effect of capsaicin on ICa. A, original current traces showing that 10 µM I-RTX abolished the effect of 1 µM capsaicin on Ica in a DRG cell. B, summary data showing lack of effect of 1 µM capsaicin on ICa at different test potentials in the presence of 10 µM I-RTX in 13 IB4-positive DRG neurons. C, mean ICa density before and after application of 1 µM capsaicin at different test potentials in nine IB4-negative but capsaicin-sensitive DRG neurons. The peak current was divided by cell capacitance to yield the current density. Data are presented in mean ± S.E. Caps, capsaicin. *, p < 0.05 compared with corresponding values before capsaicin application.

 



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  		FIG. 3.
Effect of capsaicin on four subtypes of HVACC currents. A, representative current traces showing the original recording and time course of the effect of 1 µM capsaicin on pharmacologically isolated R-, L-, P/Q-, and N-type Ca2+ currents in separate capsaicin-sensitive DRG neurons. Caps, capsaicin; con, control; Cd, cadmium; Nim, nimodipine; IVA, {omega}-agatoxin IVA; GVIA, {omega}-conotoxin GVIA; MVIIC, {omega}-conotoxin MVIIC. B, summary data showing the inhibitory effect of 1 µM capsaicin on the total and different subtypes of ICa in capsaicin-sensitive neurons. Percentage inhibition was calculated as the ratio of capsaicin-inhibited currents to either the total or the isolated subtype ICa before capsaicin application. The number of cells tested in each group is indicated in columns. Data are presented as mean ± S.E.

 



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  		FIG. 4.
Effect of capsaicin on voltage dependence activation and steady-state inactivation of ICa. A, the voltage dependence activation curves of ICa before and after 1 µM capsaicin application in 15 capsaicin-sensitive neurons. Before capsaicin application, the V0.5 and slope factor were -7.1 ± 0.2 and 5.0 ± 0.2 mV, respectively. After capsaicin, the V0.5 and slope factor were -7.8 ± 0.2 and 5.0 ± 0.1 mV, respectively. Cells were depolarized to a series of depolarizing potentials (from -60 to 20 mV with 10-mV increments) from the holding potential of -90 mV. B, representative current traces and summary data showing steady-state inactivation of ICa before and after 1 µM capsaicin in 13 capsaicin-sensitive neurons. The V0.5 was -38.9 ± 2.4 and -45.9 ± 2.5 mV (p < 0.05) before and after capsaicin application, respectively. The slope factor was -22.5 ± 3.4 and -17.9 ± 3.0 mV (p < 0.05) before and after capsaicin application, respectively. Cells were depolarized at a series of prepulse potentials (from -90 to 10 mV, 500 ms) and then depolarized to 0 mV. The normalized conductance (G/Gmax) was fit to a Boltzmann's function. Data are presented in mean ± S.E. *, p < 0.05 compared with corresponding values before capsaicin application.

 
Capsaicin Increases Steady-state Inactivation of ICa—To further characterize the inhibitory effect of capsaicin on ICa, we examined the effect of capsaicin on steady-state activation and inactivation kinetics of ICa, illustrated in Fig. 4. Although 1 µM capsaicin caused a pronounced inhibition of ICa, it did not significantly alter the voltage dependence of activation (Fig. 4A, n = 15). The decay phase of ICa was fitted with one exponential function, and the decay time constant of ICa was significantly reduced by 1 µM capsaicin (from 51.6 ± 3.0 to 39.9 ± 3.3 ms, p < 0.05; Fig. 4B; n = 15). In 13 additional capsaicin-sensitive neurons, the steady-state inactivation of ICa was examined using a series of prepulse potentials (-100 to 20 mV for 500 ms) followed by depolarizing the cell to 0 mV for 150 ms. Following application of 1 µM capsaicin, the voltage-dependent steady-state inactivation of HVACC currents was shifted significantly to the left (more negative potentials) (Fig. 4B).

Both Extracellular and Intracellular Ca2+ Are Required for the Effect of Capsaicin on ICa—The TRPV1 channel is highly permeable to Ca2+ (1). Because the degree of capsaicin-produced inhibition of ICa was proportional to the amplitude of capsaicin currents (14), we determined the role of Ca2+ influx in the inhibitory effect of capsaicin on ICa. BAPTA, a rapid Ca2+ chelator (30), was included in the pipette solution. Following the membrane rupture to achieve the whole-cell configuration, the intracellular solution exchange was allowed for 5 min before examining the effect of capsaicin on ICa. In 11 capsaicin-sensitive neurons tested, 1 µM capsaicin had no significant effect on ICa when 10 mM BAPTA was included in the pipette solution (Fig. 5A). Capsaicin also failed to alter the inactivation kinetics of ICa (Fig. 5B). Notably, ICa was activated at about -30 mV and reached its peak at 10 mV in the presence of 10 mM BAPTA. Compared with the peak ICa density and I-V curve recorded in the absence of BAPTA (95.4 ± 10.7 pA/pF at 0 mV, n = 15), intracellular dialysis of BAPTA significantly shifted the I-V curve to the right (more positive potentials) and increased the density of ICa (164.3 ± 22.2 pA/pF at 10 mV, n = 11; Fig. 5A).



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  		FIG. 5.
Effect of BAPTA on ICa and capsaicin-induced inhibition of ICa. A, original current traces and summary data showing lack of effect of 1 µM capsaicin on ICa at different test potentials in 11 capsaicin-sensitive neurons recorded with 10 mM BAPTA internal solution. The peak current was divided by cell capacitance to yield the current density. Note that ICa was activated at about -30 mV and reached its peak at 10 mV in the presence of BAPTA. B, representative current traces and summary data showing steady-state inactivation of ICa recorded with 10 mM BAPTA (from -90 to 10 mV, 500 ms) before and after capsaicin (n = 11). Before capsaicin, the V0.5 and slope factor were -35.7 ± 0.7 and -11.9 ± 0.7 mV, respectively. After capsaicin, the V0.5 and slope factor were -31.3 ± 0.9 and -14.4 ± 0.9 mV, respectively. Data are presented as mean ± S.E.

 
TRPV1 is present on both the cell membrane and endoplasmic reticulum in the DRG (31, 32). A rapid rise in intracellular Ca2+ levels may be due to either Ca2+ influx or release of Ca2+ from an intracellular store. In 12 capsaicin-sensitive DRG neurons, we replaced Ca2+ with Ba2+ in the extracellular solution to determine the role of extracellular Ca2+ in the effect of capsaicin on HVACCs. IBa was elicited at -40 mV and reached its peak at -10 mV (Fig. 6A). In the Ca2+-free external solution, 1 µM capsaicin still significantly inhibited IBa, although its inhibitory effect on IBa was significantly attenuated (37.5 ± 5.0% at -10 mV, n = 6; Fig. 6A). However, the capsaicin effect can be completely washed out in 3 min. Capsaicin did not significantly alter the steady-state inactivation kinetics of IBa (Fig. 6B).



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  		FIG. 6.
Contribution of extracellular and intracellular Ca2+ to capsaicin-induced inhibition of Ca2+ currents. A, representative tracings and summary data showing the inhibitory effect of 1 µM capsaicin on IBa (n = 12). B, capsaicin had no significant effect on the steady-state inactivation of IBa (n = 6). The V0.5 and slope factor before capsaicin were -54.5 ± 1.3 and -17.4 ± 1.6 mV, respectively. After capsaicin, the V0.5 and slope factor were -52.7 ± 1.5 and -16.4 ± 1.6 mV, respectively. C, original current traces and summary data showing lack of inhibitory effect of capsaicin on IBa in 15 DRG neurons treated with 5 µM thapsigargin. D, original recordings and summary data showing the inhibitory effect of 30 mM caffeine on IBa in 12 DRG neurons. Data are presented in mean ± S.E. *, p < 0.05 compared with corresponding values before capsaicin application.

 
We subsequently determined whether the remaining inhibitory effect of capsaicin on IBa was mediated by Ca2+ released from the intracellular stores, such as the endoplasmic reticulum. The intracellular Ca2+ store was depleted with thapsigargin, a specific Ca2+-ATPase inhibitor (33). In a total of 15 capsaicin-sensitive neurons, we pretreated the DRG cells with 5 µM thapsigargin for 10 min and washed it out for at least another 3 min using Ba2+ external solution before testing the effect of 1 µM capsaicin on IBa. The inhibitory effect of capsaicin on IBa was completely blocked in all 15 neurons treated with thapsigargin (Fig. 6C).

Furthermore, to determine whether Ca2+ release from the endoplasmic reticulum independent of TRPV1 activation can inhibit HVACCs, we examined the effect of caffeine, a selective activator for endoplasmic reticulum ryannodine receptors (34, 35), on IBa in 12 additional DRG cells. Bath perfusion of 30 mM caffeine for 1–2 min caused a significant and reversible reduction of IBa (23.7 ± 3.2% inhibition at -10 mV) (Fig. 6D).

Calmodulin and Ca2+/Calmodulin-dependent Protein Kinase II Are Not Involved in the Effect of Capsaicin on ICa—Because the increase in intracellular Ca2+ appeared essential for the inhibitory effect of capsaicin on VACCs, we next determined if calmodulin and Ca2+/calmodulin-dependent protein kinase II are involved in this effect. The specific calmodulin antagonist W-7 (36) or the selective Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93 (37) was included in the pipette solution. In the presence of W-7 (200 µM, n = 7) or KN-93 (50 µM, n = 7), 1 µM capsaicin still produced a large inhibition of ICa, which was not significantly different from the effect of capsaicin on ICa recorded using regular pipette solution without W-7 and KN-93 (Fig. 7A). W-7 and KN-93 alone did not result in inhibition of capsaicin current. Furthermore, intracellular application of a higher concentration of W-7 (500 µM, n = 4) or KN-93 (100 µM, n = 4) also failed to attenuate the inhibitory effect of 1 µM capsaicin on ICa in additional DRG neurons tested (data not shown).

Endogenous Protein Phosphatases Play a Critical Role in Regulation of HVACCs and Capsaicin-induced Inhibition of ICa—We tested the effect of okadaic acid, a specific inhibitor for protein phosphatase 1 and protein phosphatase 2A (38), on capsaicin-induced inhibition of ICa. Compared with the I-V relationship of ICa using regular pipette solution, intracellular dialysis of 0.5 µM okadaic acid significantly increased the density of ICa (193.8 ± 20.7 pA/pF at 10 mV, n = 6) and shifted the I-V curve to the right (Fig. 7B). However, inclusion of 0.5 µM okadaic acid in the pipette solution failed to attenuate the inhibitory effect of 1 µM capsaicin on ICa in all six capsaicin-sensitive neurons tested (Fig. 7B).



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  		FIG. 7.
A, representative tracings and summary data showing that intracellular application of W-7 (200 µM, n = 7) or KN-93 (50 µM, n = 7) failed to attenuate the inhibitory effect of 1 µM capsaicin on ICa. Percentage inhibition was calculated as the ratio of capsaicin-inhibited currents to the total peak current during the control. B, representative current traces and summary data showing that intracellular dialysis with 0.5 µM okadaic acid failed to block the inhibitory effect of 1 µM capsaicin on ICa at different test potentials (from -70 to 50 mV for 150 ms, n = 6). Note the I-V current shift and increased ICa density by okadaic acid. Data were presented in mean ± S.E. *, p < 0.05 compared with corresponding values before capsaicin application.

 
Calcineurin (protein phosphatase 2B) is a Ca2+-sensitive protein phosphatase and can be activated by a rise in intracellular Ca2+. To determine if capsaicin inhibits ICa through dephosphorylation mediated by calcineurin, 1 µM deltamethrin, a specific inhibitor of calcineurin (20), was included in the pipette solution. Deltamethrin completely blocked the inhibitory effect of 1 µM capsaicin on ICa in all 10 DRG neurons tested (Fig. 8, A and B). Similarly, intracellular dialysis with cyclosporin A (50 µM), another selective calcineurin inhibitor (39), also abolished the effect of capsaicin on ICa in another 10 DRG neurons examined (Fig. 8C). In the presence of deltamethrin or cyclosporin A, 1 µM capsaicin failed to alter the inactivation kinetics of ICa (Fig. 8, B and D). In separate DRG neurons, intracellular application of a lower concentration of deltamethrin (0.5 µM, n = 13) or cyclosporin A (20 µM, n = 10) only partially blocked the inhibitory effect of 1 µM capsaicin on ICa. Rapamycin binds to FK506-binding protein but does not inhibit calcineurin, because it cannot interact with the CNB subunit for steric reasons (40). In five separate DRG neurons, we found that intracellular dialysis of 20 µM rapamycin did not significantly alter the effect of capsaicin on ICa (data not shown).

Unexpectedly, inclusion of 1 µM deltamethrin alone significantly increased the density of ICa (175.9 ± 13.4 pA/pF at 10 mV, n = 10) and shifted the I-V curve of ICa to more positive potentials by ~10 mV (Fig. 8A), compared with the ICa recorded with the regular pipette solution. Similar effects on the ICa density (185.5 ± 29.3 pA/pF at 10 mV, n = 10) and the I-V curve were observed with intracellular dialysis of 50 µM cyclosporin A (Fig. 8C).

Capsaicin Induces Internalization of CaV2.2—Because capsaicin caused a profound and long lasting decrease in ICa density, the pronounced inhibition of VACCs by capsaicin exposure cannot be fully explained with increased inactivation of HVACCs. Thus, we determined whether redistribution (e.g. internalization) of HVACCs plays a role in the effect of capsaicin on ICa in DRG cells. Because the N-type Ca2+ current (mediated by CaV2.2 channels) is the most prominent subtype of HVACC currents, we chose to examine the effect of 1–10 µM capsaicin on the spatial distribution of CaV2.2 in DRG cells. The CaV2.2 distribution in TRPV1- and IB4-positive DRG neurons was studied using double immunofluorescence labeling and confocal microscopy. All negative controls (omitting primary antibodies) displayed no detectable staining. In vehicle-treated DRG neurons, the cross-sectional confocal images of CaV2.2 immunoreactivity showed a bright staining pattern with a clear and well defined CaV2.2 immunoreactivity on the plasma membrane (Fig. 9). By contrast, the cells treated with 1–10 µM capsaicin for 20 s and 2 min displayed a more diffuse and duller immunoreactivity of CaV2.2 in the cytoplasm near the cell membrane surface (Fig. 9). The inward spread of CaV2.2 immunoreactivity was more evident when compared with the labeling of TRPV1 and IB4 present in the same DRG cell (Fig. 9). Furthermore, in DRG cells pretreated with either 10 µM iodoresiniferatoxin or 1 µM deltamethrin, the spatial distribution of CaV2.2 immunoreactivity on the cell membranes was not altered by capsaicin (data not shown). In DRG cells exposed to 1–10 µM capsaicin for 3–5 min, CaV2.2 immunoreactivity completely disappeared from the plasma membrane. The cell membrane also became disintegrated following prolonged exposure to capsaicin, suggesting permanent damage to the cells (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the signaling mechanism underlying down-regulation of HVACCs by TRPV1 stimulation in native DRG neurons. Small sized DRG neurons are richly endorsed with TRPV1 (41, 42). Application of capsaicin produced a profound and sustained suppression of HVACC currents in DRG cells. We found that this inhibition was only limited to capsaicin-sensitive DRG neurons, and iodoresiniferatoxin, a highly specific TRPV1 antagonist, eliminated the effect of capsaicin. Hence, capsaicin inhibits HVACC currents specifically through TRPV1 in DRG neurons. Furthermore, we observed that capsaicin produced a similar degree of inhibition in all the subtypes (N-, P/Q-, L-, and R-type) of HVACCs. This suggests that a common mechanism is probably responsible for the effect of capsaicin on different subtypes of HVACCs. Although capsaicin had no effect on the voltage dependence activation of HVACCs, it increased the steady-state inactivation of HVACC currents. Thus, the inhibitory effect of capsaicin on HVACC currents appears to be due in part to a decrease in the open state probability of HVACCs in DRG neurons.

What then are the downstream signaling mechanisms responsible for the down-regulation of HVACCs by capsaicin in DRG neurons? Stimulation of G protein-coupled receptors inhibits HVACCs through G protein {beta}{gamma}-subunits (25, 26). However, we obtained no evidence that G proteins are involved in the effect of capsaicin on HVACCs. This is because depolarizing pulses did not lead to a prepulse-induced facilitation (relief of inhibition) of Ca2+ currents. Furthermore, pretreatment of DRG neurons with pertussis toxin to inactivate inhibitory Gi/o proteins had no effect on capsaicin-induced inhibition on HVACC currents. Thus, the G proteins are not involved in the rapid inhibition of HVACCs by capsaicin.



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  		FIG. 8.
Effect of calcineurin inhibitors on ICa and capsaicin-induced inhibition of ICa. A, representative current traces and summary data showing lack of effect of 1 µM capsaicin on ICa at different test potentials (from -70 to 50 mV for 150 ms) following intracellular dialysis with 1 µM deltamethrin (n = 10). Note to the right of the I-V curve of ICa, the increased ICa density in the presence of deltamethrin. B, original current traces and summary data showing that capsaicin failed to alter the steady-state inactivation curve of ICa following intracellular application of deltamethrin (n = 5). The V0.5 and slope factor before capsaicin application were -50.4 ± 1.9 and -19.3 ± 2.0 mV, respectively. After capsaicin, the V0.5 and slope factor were -46.7 ± 1.8 and -15.9 ± 2.0 mV, respectively. C, representative current traces and summary data showing a lack of inhibitory effect of 1 µM capsaicin on ICa at different test potentials (from -70 to 50 mV) following intracellular dialysis with 50 µM cyclosporin A (n = 10). Note the I-V current shift and increased ICa density by cyclosporin A. D, current traces and summary data showing that 1 µM capsaicin failed to shift the inactivation curve in the presence of cyclosporin A (n = 5). The V0.5 was -47.8 ± 2.0 and -46.8 ± 2.7 mV before and after capsaicin application, respectively. The slope factor was -20.5 ± 2.7 and -21.6 ± 3.4 mV before and capsaicin application, respectively.

 
Because TRPV1 is highly permeable to Ca2+ (1, 2), capsaicin can induce substantial Ca2+ influx into the DRG cell. In our study, intracellular dialysis with BAPTA, a rapid Ca2+ chelator (30), abolished the inhibitory effect of capsaicin on Ca2+ currents, suggesting that the rise in intracellular Ca2+ is the key event in capsaicin-induced inhibition of HVACC currents. Although these data clearly indicate the importance of intracellular Ca2+ in the effect of capsaicin on HVACCs, they do not discriminate the sources of Ca2+. Capsaicin can activate TRPV1 located on both the plasma membrane and endoplasmic reticulum (31, 32). Consequently, we examined more closely the relative contribution of intracellular and extracellular Ca2+ to the effect of capsaicin on HVACC currents. When the extracellular Ca2+ was replaced with barium, the inhibitory effect of capsaicin was significantly attenuated, and its effect was completely washed out. Furthermore, depletion of intracellular Ca2+ with thapsigargin, a highly specific inhibitor of Ca2+-ATPases (33), abolished the capsaicin-induced inhibition on HVACC currents. Thus, the inhibitory effect of capsaicin on HVACCs is triggered by both Ca2+ influx and release of Ca2+ from intracellular stores. We found that stimulation of endoplasmic reticulum ryannodine receptors with caffeine (34, 35), significantly inhibited IBa in DRG neurons. These data strongly suggest that Ca2+ release from the endoplasmic reticulum contributes to the inhibitory effect of capsaicin on HVACCs. In addition, capsaicin increases the steady-state inactivation of HVACC currents through an increase in intracellular Ca2+, which highlights a direct Ca2+-dependent inactivation of HVACCs in primary sensory neurons.

The most salient finding of this study is that calcineurin plays a pivotal role in down-regulation of HVACCs caused by TRPV1 stimulation in DRG neurons. Calcineurin is a Ca2+-dependent protein phosphatase and is enriched in the DRG neurons (43). We found that inhibition of calcineurin with intracellular dialysis of either deltamethrin or cyclosporin A, two structurally dissimilar calcineurin inhibitors, completely blocked the effect of capsaicin on HVACC currents. On the other hand, intracellular application of okadaic acid, a specific inhibitor for protein phosphatases protein phosphatase 1 and protein phosphatase 2A, failed to attenuate the effect of capsaicin. Deltamethrin and structurally related Type II pyrethroids are potent inhibitors of calcineurin (20, 44, 45). Cyclosporin A specifically binds to the intracellular receptor protein cyclophilin A, a member of the immunophilin protein family (39), and the resulting complex is an effective inhibitor of calcineurin. Interestingly, rapamycin, an inhibitor of FK506-binding protein but ineffective in inhibiting calcineurin activity (40), did not alter the effect of capsaicin on Ca2+ currents. Calcineurin may be activated by Ca2+, calmodulin, and inhibitory G proteins (46, 47). However, it appears that Ca2+ is a direct activator of calcineurin following stimulation of TRPV1 in DRG neurons, since treatment with W-7, KN-93, or pertussis toxin failed to attenuate the effect of capsaicin. These data strongly suggest that an increase in intracellular Ca2+ by TRPV1 leads to down-regulation of HVACCs by protein dephosphorylation mediated by Ca2+-dependent calcineurin.

We also demonstrate for the first time that stimulation of TRPV1 causes a rapid internalization of HVACCs in DRG neurons. Using double immunofluorescence labeling and confocal microscopy, we found that capsaicin induced a rapid internalization of CaV2.2 immunoreactivity in IB4- and TRPV1-positive DRG neurons. Since capsaicin caused a similar degree of down-regulation in all four subtypes of HVACCs, it is probable that internalization occurs in the other three subtypes of HVACCs. It is not clear if the substrate of calcineurin is the HVACCs or a regulatory phosphoprotein that interacts with the channel in the DRG. We found that capsaicin shifted the reversal potential of HVACCs, which is probably due to dephosphorylation of HVACCs, resulting in a structural change of channel proteins, through activation of Ca2+-dependent calcineurin. In addition to direct phosphorylation and dephosphorylation of HVACCs (16, 48), protein kinases and calcineurin can phosphorylate and dephosphorylate, respectively, the cytoskeletal proteins, which in turn affect the HVACC activity (49, 50). Therefore, internalization of HVACCs appears to be an important mechanism responsible for capsaicin-induced profound loss of the HVACC density in DRG neurons. Further studies are warranted to identify the exact substrates dephosphorylated by calcineurin in the DRG neurons.



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  		FIG. 9.
Representative high magnification confocal images showing the spatial distribution of CaV2.2 immunoreactivity in IB4-positive (A) and TRPV1-positive (B) DRG neurons treated with the vehicle or 1 µM capsaicin for 20 s and 2 min. Digitally merged images from CaV2.2 (green) and IB4/TRPV1 (red) labeling are shown on the right. Scale bar, 10 µm. All images are single confocal optical sections.

 
Another intriguing and important finding is that the basal intracellular Ca2+ and endogenous calcineurin tonically modulate the HVACC currents in DRG neurons. In this regard, intracellular dialysis with BAPTA, deltamethrin, or cyclosporin A all significantly increased the Ca2+ current density and induced a depolarizing shift in the voltage dependence of activation. The observation that BAPTA and calcineurin inhibitors resulted in increased density of Ca2+ currents points to a basal activity of calcineurin in DRG cells in the absence of any prior evoked increases in intracellular Ca2+. Notably, a similar effect was observed when okadaic acid was applied intracellularly, suggesting that the phosphatases protein phosphatase 1 and protein phosphatase 2A may also be involved in the basal regulation of HVACCs. Therefore, a substantial proportion of HVACCs or closely associated proteins in the DRGs are present in the dephosphorylated form in the DRG. Consistent with our findings, overexpression of calcineurin in NG108-15 cell lines causes a decreased current density of HVACCs. By contrast, the current density of HVACCs is increased in those cells transfected with the calcineurin antisense (16). HVACCs in the dynamic equilibrium of calcineurin-protein kinase activity allow DRG cells to both increase and reduce Ca2+ influx and, by this mechanism, contribute to regulation of neurotransmitter release and nociceptive inputs to spinal dorsal horn neurons.

In summary, this study provides substantial new evidence that calcineurin, constitutively expressed in the cytoplasm of DRG neurons, is a key feedback regulator of intracellular Ca2+ and plays an important role in down-regulation of HVACCs by TRPV1 stimulation. Increased calcineurin activity produced by TRPV1 activation could limit Ca2+ influx through HVACCs in the plasma membrane by down-regulation of HVACCs through dephosphorylating the HVACC or a closely associated cytoskeletal protein. This new information is important for our understanding of the molecular mechanism of the analgesic action and diminished spinal synaptic transmission produced by capsaicin and its related analogs. This study highlights the pivotal role of intracellular Ca2+ level and calcineurin in negative modulation of HVACCs in primary sensory neurons.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM64830 and NS45602. 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 Anesthesiology, H187, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-8433; Fax: 717-531-6221; E-mail: hpan{at}psu.edu.

1 The abbreviations used are: TRPV1, transient receptor potential vanilloid type 1; DRG, dorsal root ganglion; IB4, Griffonia simplicifolia isolectin B4; VACCs, voltage-activated calcium channels; HVACCs, high voltage-activated calcium channels; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulyonamide; TEA, tetraethylammonium; pF, picofarads. Back



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Y. V. Medvedeva, M.-S. Kim, and Y. M. Usachev
Mechanisms of Prolonged Presynaptic Ca2+ Signaling and Glutamate Release Induced by TRPV1 Activation in Rat Sensory Neurons
J. Neurosci., May 14, 2008; 28(20): 5295 - 5311.
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 J. Pharmacol. Exp. Ther.Home page
H.-Y. Zhou, H.-M. Zhang, S.-R. Chen, and H.-L. Pan
Increased C-Fiber Nociceptive Input Potentiates Inhibitory Glycinergic Transmission in the Spinal Dorsal Horn
J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 1000 - 1010.
[Abstract] [Full Text] [PDF]

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 JDRHome page
G. Chung, J.N. Rhee, S.J. Jung, J.S. Kim, and S.B. Oh
Modulation of CaV2.3 Calcium Channel Currents by Eugenol
Journal of Dental Research, February 1, 2008; 87(2): 137 - 141.
[Abstract] [Full Text] [PDF]

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T. Fischbach, W. Greffrath, H. Nawrath, and R.-D. Treede
Effects of Anandamide and Noxious Heat on Intracellular Calcium Concentration in Nociceptive DRG Neurons of Rats
J Neurophysiol, August 1, 2007; 98(2): 929 - 938.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
Z.-Z. Wu and H.-L. Pan
Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R276 - R283.
[Abstract] [Full Text] [PDF]

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C.-K. Park, H.Y. Li, K.-Y. Yeon, S.J. Jung, S.-Y. Choi, S.J. Lee, S. Lee, K. Park, J.S. Kim, and S.B. Oh
Eugenol Inhibits Sodium Currents in Dental Afferent Neurons
Journal of Dental Research, October 1, 2006; 85(10): 900 - 904.
[Abstract] [Full Text] [PDF]

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S. R. Kim, S. U. Kim, U. Oh, and B. K. Jin
Transient Receptor Potential Vanilloid Subtype 1 Mediates Microglial Cell Death In Vivo and In Vitro via Ca2+-Mediated Mitochondrial Damage and Cytochrome c Release
J. Immunol., October 1, 2006; 177(7): 4322 - 4329.
[Abstract] [Full Text] [PDF]

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 J. Neurophysiol.Home page
S.-R. Chen and H.-L. Pan
Loss of TRPV1-Expressing Sensory Neurons Reduces Spinal {micro} Opioid Receptors But Paradoxically Potentiates Opioid Analgesia
J Neurophysiol, May 1, 2006; 95(5): 3086 - 3096.
[Abstract] [Full Text] [PDF]

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 Mol. Biol. CellHome page
J. Hu, Y.-K. Bae, K. M. Knobel, and M. M. Barr
Casein Kinase II and Calcineurin Modulate TRPP Function and Ciliary Localization
Mol. Biol. Cell, May 1, 2006; 17(5): 2200 - 2211.
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