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Originally published In Press as doi:10.1074/jbc.M605248200 on December 4, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4757-4764, February 16, 2007
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Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons*

Zhi Fang{ddagger}1, Chul-Kyu Park{ddagger}1, Hai Ying Li{ddagger}, Hyun Yeong Kim{ddagger}, Seong-Hae Park{ddagger}, Sung Jun Jung§, Joong Soo Kim{ddagger}, Arnaud Monteil, Seog Bae Oh{ddagger}2, and Richard J. Miller||

From the {ddagger}Department of Physiology and Program in Molecular and Cellular Neuroscience, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, Korea, the §Department of Physiology, College of Medicine, Kangwon National University, Chunchon 200-710, Korea, the Département de Physiologie, Institut de Génomique Fonctionnelle, CNRS-UMR 5203 141, Rue de la Cardonille 34396 Montpellier Cedex 5, France, and the ||Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611

Received for publication, June 1, 2006 , and in revised form, November 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cav2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Cav2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II–III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Cav2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Cav2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Cav2.3a and Cav2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Cav2.3e is the major isoform, whereas Cav2.3e expression is highly restricted to small (<16 µm) isolectin B4-negative and tyrosine kinase A-positive neurons. Cav2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Cav2.3e expression. Our results suggest that Cav2.3e in sensory neurons may be a potential target for the treatment of pain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The trigeminal ganglion (TG)3 neurons are involved in the transmission of orofacial sensory information, including pain (1). Physical, chemical, and inflammatory damage in peripheral tissues gives rise to increased excitability of nociceptive neurons (2). Calcium plays a key role in cellular processes under these conditions, and the major routes for calcium entry into the cell are the voltage-activated calcium channels (VACCs) (3, 4). Indeed, VACCs have a variety of physiological functions, including regulation of firing patterns, synaptic modulation, and neurotransmitter release in the nervous system (5).

VACCs are formed by one of several pore-forming {alpha}1 subunits ({alpha}1A-I and {alpha}1S) and auxiliary subunits. Molecular characterizations have determined that {alpha}1C, {alpha}1D, {alpha}1F, and {alpha}1S subunits encode L-type (Cav1.1–1.4) Ca2+ channels (6, 7); {alpha}1A encodes P/Q-type (Cav2.1) channels (8); {alpha}1B encodes N-type (Cav2.2) channels (9); {alpha}1E encodes R-type (Cav2.3) channels (10); and {alpha}1G, {alpha}1H, and {alpha}1I encode T-type (Cav3.1–3.3) channels (11, 12). R-type currents with diverse biophysical properties were described in different types of neurons in the central nervous system (13, 14). Although the molecular nature of R-type currents in neurons is not fully understood (15, 16), to date, six Cav2.3 spliced variants (Cav2.3a to Cav2.3f) have been described in various mammalian species (14, 17–20).

In primary sensory neurons, Cav2.3 calcium channels have been suggested to contribute to pain transmission (21). Immunohistochemical and in situ hybridization analysis showed heterogeneous expression of Cav2.3 calcium channels in sensory neurons (22, 23). Electrophysiological studies also demonstrated the presence of R-type currents in subsets of sensory neurons that result from Cav2.3 expression (15, 24). However, Cav2.3 calcium channel isoforms expressed in sensory neurons have not been characterized.

In this study, we therefore determined expression patterns of Cav2.3 isoforms in the rat TG nociceptive neurons by the combination of molecular and functional analyses. Nociceptive TG neurons were identified by size and the expression of isolectin B4 (IB4)-binding protein, tyrosine kinase A (trkA), and transient receptor potential vanilloid 1 (TRPV1). As a result, we found that Cav2.3e is the major Cav2.3 isoform in TG neurons, and Cav2.3e is preferentially expressed in small (<16 µm) IB4-negative/trkA-positive and TRPV1-positive neurons. In addition, we demonstrate that SNX-482-sensitive R-type calcium channel component is associated with Cav2.3e expression in these neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of TG Neurons—All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the School of Dentistry, Seoul National University. TG neurons from 2- to 5-day-old neonatal rats were prepared as described previously (25). Briefly, TG neurons were washed several times in cold (4 °C) Hanks' balanced salt solution (Invitrogen) and then incubated for 20 min at 37 °C in Hanks' balanced salt solution containing trypsin. The cells were washed in Dulbecco's modified Eagle's medium and triturated with a flame-polished Pasteur pipette to separate cells and remove processes. Subsequently, cells were centrifuged, resuspended, and placed on poly-L-ornithine (0.5 mg/ml)-coated glass coverslips (25 mm in diameter). Cells were maintained in an incubator at 37 °C equilibrated with 5% CO2.

Immunocytochemistry—For immunocytochemistry, trigeminal ganglion neurons were seeded on poly-L-ornithine-coated coverglass and maintained in a 5% CO2 incubator at 37 °C. The TG neurons were used for the experiments at 1 day after culture. After rinsing in 0.1 M phosphate-buffered saline (PBS), the TG neurons were fixed in -20 °C methanol (Merck) for 10 min. Afterward, the TG neurons were preincubated in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 1 h at room temperature and then incubated in rabbit anti-Cav2.3 ({alpha}1E voltage-gated calcium channel, R-type) (1:200; Chemicon) in the same solution at 4 °C overnight. The cells were washed three times with PBS and then incubated with FITC-conjugated rabbit IgG antibody (1:200; Jackson Immuno-Research) for 1 h at room temperature. After washing with PBS, the samples were covered with VectaShield mounting (Vector Laboratories), and fluorescent images were obtained under a confocal microscope (FV-300, Olympus, Japan). The specificity of primary antibody was confirmed by control antigen for anti-Cav2.3. For negative control, 1 µg of blocking peptide was preincubated with the same volume of primary antibody for 1 h at room temperature.

Whole Tissue RT-PCR Analysis—Total RNA was isolated from 2- to 5-day-old rats TG neurons using the TRIzol® reagent (Invitrogen). Following digestion with DNase I, 3 µg of total RNA was used for cDNA synthesis with the SuperscriptTM first-strand synthesis system (Invitrogen) according to the manufacturer's instructions. After the reverse transcription reaction, 1 ng of cDNA was then used as a template for amplification. Primers for PCR were specifically designed to differentiate the presence or the absence of three inserts (inserts I, II, and III) in the Cav2.3 transcripts based on GenBankTM rat cDNA sequences (Table 1). After a denaturation step of 5 min at 94 °C, the amplification was carried out at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s for 35 cycles. The PCR was completed by maintaining temperatures at 72 °C for 10 min. As a positive control, cDNA from the same preparations was subjected to 35 cycles of PCR with primers for beta-actin. All PCR products were resolved on 2% agarose gels.


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  		TABLE 1
List of primer sequences designed for this study

 
Single Cell RT-PCR—We adopted methods described by Silbert et al. (26) for single cell RT-PCR. An entire single cell or the intracellular content of a single cell (when performed just after electrophysiological recording) was aspirated into a patch pipette using negative pressure under visual control. Patch pipettes used for an entire neuron harvest had a tip diameter range of 12–30 µm and were filled with RNase-free water. The tip of the pipette and its contents were broken into a reaction tube containing RT reagents. To avoid genomic DNA contamination, digestion with DNase I was performed before RT. RT was carried out for 1 h at 50 °C (Invitrogen), and the cDNA product was used in a separate PCR. The forward and reverse primers were designed from unique sequence to avoid amplifying homologous genes. The first round of PCR was performed in 50 µl of PCR buffer containing 0.2 mM dNTPs, 0.2 µM"outer" primers, 5 µl of RT product, and 0.2 µl of platinum Taq DNA polymerase (Invitrogen). The protocol included a 5-min initial denaturation step at 95 °C followed by 60 cycles of 40 s of denaturation at 95 °C, 40 s of annealing at 60 °C, and 40 s of elongation at 72 °C. The reaction was completed with 7 min of final elongation. For the second round of amplification, the reaction buffer (20 µl) contained 0.2 mM dNTPs, 0.2 µM"inner" primers, 5 µl of the first round PCR products, and 0.1 µl of platinum Taq DNA polymerase. "Insert" primers, designed to detect the presence of insert fragments, were also used in second round amplifications. The reaction procedure for these primers was the same as the first round. The sequences of all the primers used for single cell PCR are presented in Table 1. For positive controls, beta-actin primers were used in parallel PCRs. A negative control was obtained from pipettes that did not harvest any cell contents but were submerged in the bath solution. The PCR products were displayed on ethidium bromide-stained 2% agarose gels. Gels were photographed using a digital camera (Bioprint 2000 x-press zoom, Vilber Lourmat, France).

Classification of Sensory Neurons—As described previously (27), TG neurons were classified into three groups as follows: small (10–16 µm), medium (16–20 µm), and large (20–30 µm) neurons. Griffonia simplicifolia IB4 was also utilized to classify TG neurons as either IB4-positive or IB4-negative neurons (28). Before single cell collections, TG neurons were incubated with 10 µg/ml IB4-FITC (Sigma) in a balanced salt solution (BSS (in mM) as follows: 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose) for 10 min and then rinsed in BSS. For electrophysiological experiments, neurons were stained with 10 µg/ml IB4-FITC for 10 min and then rinsed for 10 min in extracellular solution before recording. IB4-FITC staining was visualized with standard FITC filters.

Intracellular Calcium Imaging—Intracellular calcium imaging was performed as described previously (25). Briefly, neurons were loaded with fura-2 AM (2 µM; Molecular Probes, Eugene, OR) for 40 min at 37 °C in BSS. The cells were then rinsed and incubated for 30 min to de-esterify the dye. The cells were plated onto poly-L-ornithine-coated coverslips, mounted onto the chamber, placed on an inverted microscope (Olympus IX70, Japan) and perfused continuously with BSS at 2 ml/min. All measurements were made at 36 °C (temperature controller PTC-20; ALA Scientific Instrument Inc.). Cells were illuminated with a 175-watt xenon arc lamp, and excitation wavelengths (340/380 nm) were selected by a Lambda DG-4 monochromator wavelength changer (Shutter Instrument, Novato, CA). Intracellular free calcium concentration ([Ca2+]i) was measured by digital video microfluorometry with an intensified CCD camera (CasCade, Roper Scientific, Trenton, NJ) coupled to a microscope and software (Metafluor, Universal Imaging Corp., Downingtown, PA) on a Pentium 4 computer.

Electrophysiological Recordings—We performed whole-cell patch clamp recordings to measure barium currents (IBa) with an Axopatch-1C amplifier (Axon Instruments, Union City, CA). The pipette resistance was 2–5 megohms. Series resistance was compensated for (>80%), and leak subtraction was performed. Data were low pass-filtered at 2 kHz and sampled at 10 kHz. The pClamp8 (Axon Instruments) software was used during experiments and analysis. The pipette solution for IBa contained the following (mM): 100 CsCl, 1 MgCl2, 10 HEPES, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 3.6 Mg-ATP, 14 phosphocreatine, 0.1 GTP, and 50 units/ml creatine phosphokinase, adjusted to pH 7.4 with CsOH. The extracellular solution for IBa contained the following (mM): 151 tetraethylammonium chloride, 10 HEPES, 5 BaCl2, 1 MgCl2, and 10 glucose, adjusted to pH 7.4 with tetraethylammonium OH. The IBa was evoked by a test pulse to +0 mV from the holding potential, -80 mV every 10 s.


Figure 1
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  		FIGURE 1.
The representative photographs showing immunoreactivity of Cav2.3 in TG neurons. A, TG neurons visualized under FITC filter (panel a) and overlay with differential interference contrast (panel b). Cells with Cav2.3 immunoreactivity are shown by arrows; cells with no Cav2.3-immunoreactivity are shown by arrowheads. The expression of Cav2.3 is predominant in small TG neurons. B, TG neurons following preabsorption of the Cav2.3 primary antibody with its blocking peptide. Panel a, under FITC filter; panel b, differential interference contrast. The scale bar indicates 20 µm.

 
Drugs—Nimodipine (Sigma) was dissolved in methanol. {omega}-Conotoxin-GVIA ({omega}-CgTx), {omega}-agatoxin-IVA ({omega}-Aga; Alomone Labs, Jerusalem, Israel), SNX-482, and cadmium chloride (Sigma) were dissolved in distilled water to make a stock solution and kept at -20 °C. The drugs were diluted to the final concentration in the extracellular solution and applied by gravity through the bath perfusion system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cav2.3 Is Heterologously Expressed in Sensory Neurons—The distribution of Cav2.3 was determined by immunocytochemical approach in rat TG neurons that were prepared from primary culture. We found that Cav2.3 was not evenly distributed in the cell bodies of TG neurons. As shown in Fig. 1, the staining of Cav2.3 in TG neurons was heterogeneous with different levels of immunoreactivity, being predominantly expressed in small sensory neurons than large neurons. These results suggest the potential role of Cav2.3 in nociceptive neurotransmission. Thus, we further examined which isoforms of Cav2.3 are specifically expressed in nociceptive sensory neurons.


Figure 2
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  		FIGURE 2.
A, putative membrane topology of the Cav2.3 subunit. The structural variations include 19 (insert I) and 7 (insert II) amino acid (aa) segments in the loop between domains II and III and a 43-amino acid segment (insert III) in the proximal carboxyl terminus. B, eight possible isoforms deduced from the Cav2.3 sequence. Cav2.3a-Cav2.3f is a newly proposed set of names by Pereverzev et al. (20). The isoform names in parentheses are initial names. C, the illustration shows the locations in Cav2.3 subunit of the primers designed for RT-PCR analysis.

 
Two Cav2.3 Isoforms, Cav2.3a and Cav2.3e, Are Expressed in Sensory Neurons—The presence of three major inserts was investigated to determine splice variants of Cav2.3. The inserts tested were I and II in the II–III loop and III in the carboxyl terminus (Fig. 2A). For RT-PCR, we designed primers flanking each insert (named inner primers) as well as on the insert region (named insert primers) (see Table 1 and Fig. 2C). Using inner primers, the PCR products were differentiated by size, because amplification with the insert yielded products of larger size than amplification without the insert. Cav2.3 containing the insert was further verified using insert primers. One of the insert primers was within the insert region, although the other was outside the region (Fig. 2C). In this scenario we only obtained amplification when the insert was present. As a result, we only found amplification of insert II in the II–III loop. In the carboxyl terminus, however, we obtained both short and long cDNA fragments (absence and presence of the 129-bp insert III fragment) (Fig. 3A). These two isoforms correspond to Cav2.3a and Cav2.3e, respectively (Fig. 3B).

Cav2.3e Is the Major Cav2.3 Isoform in Sensory Neurons— From whole tissue RT-PCR analysis, we found that the Cav2.3a and Cav2.3e isoforms are expressed in TG neurons. We further determined the expression pattern of these two isoforms at the single cell level. When we analyzed inserts I, II, and III, using single cell RT-PCR, the splicing patterns were consistent with those obtained in whole tissue. We detected the Cav2.3a and Cav2.3e isoforms in a subpopulation of TG neurons (Fig. 3C). In further experiments, we only analyzed insert III to discriminate between Cav2.3 isoforms in individual neurons (n = 78, chosen irrespective of size). Of the neurons analyzed, Cav2.3e and Cav2.3a mRNAs were found in 19.2% (n = 15/78) and 2.5% (n = 2/78), respectively (Fig. 3D). We failed to observe the expression of Cav2.3e and Cav2.3a expressed together in the same TG neuron. Our data demonstrate that Cav2.3e is the major Cav2.3 isoform in rat TG neurons.

Expression of Cav2.3e Isoform Is Predominant in Small Sensory Neurons—The observation that Cav2.3e is the major isoform in TG neurons led us to analyze only Cav2.3e in subsequent single cell RT-PCR analyses. TG neurons were categorized into small (10–16 µm), medium (16–20 µm), and large (20–30 µm) neurons, as described previously (27). Cav2.3e mRNA was detected in 30.9% (n = 13/42) of small neurons and 11.1% (n = 2/18) of medium neurons but was absent from the large neurons (n = 0/18) (Fig. 4A).

Preferential expression of Cav2.3e in small neurons suggests that Cav2.3e might be involved in nociception (29). Small diameter sensory neurons can be divided into two groups based on their neurochemical properties (30). One group contains neuropeptides such as calcitonin gene-related neuropeptide and substance P and expresses the high affinity nerve growth factor receptor trkA. The other group lacks neuropeptides but binds IB4 and expresses P2X3 (31–33). Therefore, we further characterized the expression of Cav2.3e in both groups. In agreement with the previous report (30), the majority of IB4-positive and IB4-negative neurons expressed P2X3 mRNA (n = 10/12) and trkA mRNA (n = 24/30), respectively. Cav2.3e mRNA was not detected in IB4-positive/P2X3-positive neurons (n = 0/10), but it was detected in IB4-negative/trkA-positive neurons (n = 13/24) (Fig. 4B).

The TRPV1 receptor, a member of the vanilloid receptor subfamily of the transient receptor potential channel superfamily, is a well known nociceptive marker in sensory neurons (34, 35). We examined the correlation between Cav2.3e mRNA expression and TRPV1 mRNA in small TG neurons. 76.9% (n = 10/13) of neurons expressing Cav2.3e were TRPV1-positive (Fig. 4C). Taken together, these results indicate that Cav2.3e is restricted to trkA-positive, IB4-negative neurons and is preferentially expressed in TRPV1-positive small TG neurons.


Figure 3
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  		FIGURE 3.
Two Cav2.3 isoforms were detected in TG neurons. A, inserts I–III were analyzed by whole tissue RT-PCR. The expected products sizes are as follows. Inner primer, {Delta}insert I (203 bp) and +insert I (260 bp); {Delta}insert II (259 bp) and +insert II (280 bp); {Delta}insert III (138 bp) and +insert III (267 bp); Insert primer, {Delta}insert I (no product) and +insert I (378 bp); {Delta}insert II (no product), and +insert II (392 bp); {Delta}insert III (no product) and +insert III (307 bp). The PCR products from TG neurons never have insert I, but always have insert II and either lack or contain insert III. B, illustrated are representative gels showing single cell RT-PCR products amplified using specific primers. Cav2.3 isoform that has insert II, but no insert I and III, corresponds to Cav2.3a. Cav2.3 isoform that has insert II and III, but no insert I, corresponds to Cav2.3e. C, single cell RT-PCR products amplified with nested primers from individual neurons were the same as PCR products from whole tissue, i.e. missing insert I, containing insert II, and either containing (lane 1) or missing (lane 2) insert III. Note that two PCR products with or without insert III (larger, lane 1 and smaller lane 2) were produced in different cells at single cell level, indicating that Cav2.3a and Cav2.3e are not expressed together in the same cells. D, the number (lanes 1–6) indicates six different neurons examined by single cell RT-PCR. beta-Actin was used in each reaction as a positive control. This shows the proportion of TG neurons that contain Cav2.3e and Cav2.3a, respectively.

 
Cav2.3e Expression Is Associated with a Functionally Identified R-type Calcium Channel in Subsets of Sensory Neurons—Using calcium imaging and whole-cell patch clamp recording, we then examined whether Cav2.3e expression is functionally linked to the R-type calcium channel in TG neurons. High K+ (30 mM) application readily elicits large calcium transient that was not blocked with the pretreatment of 1 µM thapsigargin in the bath solution (Fig. 5B), indicating that this calcium transient results from calcium influx in TG neurons (Fig. 5A). In some neurons (n = 9/15), the residual calcium transients remained, even after applying a mixture of calcium channel blockers (Fig. 5C). Because of complete blockade by CdCl2 (200 µM), this remaining calcium transient was attributed to R-type calcium channel components (Fig. 5C). The mixture contains L-, N-, and P/Q-type Ca2+ channel blockers (500 nM nimodipine, 500 nM {omega}-CgTx, and 200 nM {omega}-AgaIVA). In the other neurons (n = 6/15), the calcium transients were completely abolished by applying the mixture (Fig. 5D). When the expression of Cav2.3e was determined by single cell RT-PCR after imaging, Cav2.3e mRNA was found in a subset of TG neurons that have an R-type calcium channel component (n = 5/9) (Fig. 5C). In contrast, Cav2.3e mRNA was not detected in neurons, with no residual calcium transient (n = 0/6) (Fig. 5D). Cav2.3a was not detected in any TG neurons tested (n = 0/15).


Figure 4
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  		FIGURE 4.
Distribution of Cav2.3e in nociceptive TG neurons. A, expression patterns of Cav2.3e were analyzed in three groups categorized according to TG neuron size (small, medium, and large). The representative gels show single cell RT-PCR products obtained from six (lanes 1–6) different neurons. Control beta-actin was used in each single cell RT-PCR. The numbers of Cav2.3e-expressing neurons over total neurons tested are presented in the bar graph. B, distribution of Cav2.3e in small diameter sensory neurons. Illustrated are representative gels showing single cell RT-PCR products obtained from six (lanes 1–6) different neurons, IB4-negative and -positive TG neurons. After small diameter neurons in TG were classified as IB4-negative or IB4-positive neurons, trkA and P2X3 expression in each group was first determined by single cell RT-PCR. Then the expression pattern of Cav2.3e in each group was examined. The graph shows that Cav2.3e isoform expression was only restricted to IB4-negative/trkA-positive sensory neurons but not in IB4-positive neurons. C, illustrated are representative gels showing RT-PCR products amplified with Cav2.3e, TRPV1, and beta-actin-specific primers from four different neurons. The graph shows that Cav2.3e is preferentially expressed in TRPV1-positive neurons.

 
We next performed whole-cell patch clamp recordings from the identified nociceptive TG neurons using IB4-FITC (Fig. 6A). In all IB4-negative neurons tested (n = 10), we observed that residual calcium currents remained even after applying a mixture solution of calcium channel blockers, and this residual current was sensitive to SNX-482 as the majority (~80%) of the residual currents was abolished by SNX-482 (Fig. 6B). Cav2.3e mRNA was found in 70% of IB4-negative neurons tested (n = 7/10) (Fig. 6B). However, in IB4-positive neurons tested (n = 13), Cav2.3e mRNA was not found regardless of the presence of residual calcium currents after applying calcium channel blockers (Fig. 6C). Furthermore, the residual currents in IB4-positive neurons were not abolished by SNX-482 (Fig. 6C, panel b). Consistent with calcium imaging results, Cav2.3a was not found in IB4-negative or IB4-positive neurons (Fig. 6, B and C). Taken together, these results suggest that Cav2.3e mRNA is associated with the generation of SNX-482-sensitive R-type calcium channel components in IB4-negative nociceptive neurons.


Figure 5
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  		FIGURE 5.
Single cell RT-PCR analysis following fura-2 based calcium imaging. A, representative traces from an individual TG neuron obtained by subsequent application of high K+ solution (30 mM KCl) (•), which produced consistent responses upon triple application. B, Ca2+ transients obtained from depolarizing the TG neurons with high K+ solution was because of Ca2+ influx via calcium channels rather than mobilization of intracellular Ca2+ store. The pretreatment of thapsigargin failed to abolish high K+-induced calcium transients (n = 12, p > 0.01). The illustrated is a representative trace from a TG neuron. C, in a subpopulation of TG neurons (n = 9/15), calcium transient still remained in the presence of mixture solution of L-, N-, and P/Q-type blockers (bar); nimodipine, 500 nM; {omega}-CgTx, 500 nM; and {omega}-AgaIVA, 200 nM. This remaining calcium transients were completely blocked by application of CdCl2 (200 µM). This is a representative experiment. Single cell RT-PCR analysis following calcium imaging revealed Cav2.3e expression in the same cell (n = 5/9) but no Cav2.3a expression (n = 9/9). D, in the other TG neurons (n = 6/15), calcium transient was completely abolished by mixture solution. Neither Cav2.3e nor Cav2.3a mRNA was detected in these neurons (n = 6/6).

 


Figure 6
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  		FIGURE 6.
Single cell RT-PCR analysis following whole-cell patch clamp recordings. A, TG neurons acutely isolated from neonatal rats were visualized under fluorescence microscope (panel a) after IB4-FITC treatment prior to recording (panel b). Panel c, overlay. Arrowheads indicate IB4-negative cell; arrows indicate IB4-positive cell. B, in IB4-negative cells tested (n = 10), residual currents resulting from R-type calcium channel components remained in the presence of L-, N- and P/Q-type blockers (bar); nimodipine, 500 nM; {omega}-CgTx, 500 nM; and {omega}-AgaIVA, 200 nM. The residual currents were further blocked by the R-type calcium channel blocker SNX-482 (100 nM), and the calcium channel blocker CdCl2 (200 µM). Single cell RT-PCR analysis following recording revealed Cav2.3e expression in the same cell (n = 7/10) but no Cav2.3a expression (n = 10/10). C, in IB4-positve cells tested (n = 13), residual currents did not remain in some cells (a, n = 6) but remained in the other cells (b, n = 7). Neither the Cav2.3e nor Cav2.3a transcript was detected in these neurons (n = 13).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
R-type calcium channels have been demonstrated to contribute to pain transmission in sensory neurons. In this study, we have identified specific isoforms of the Cav2.3 subunit expressed in TG nociceptive neurons. Two Cav2.3 isoforms, Cav2.3a and Cav2.3e, were found, with Cav2.3e being the predominant form. The Cav2.3e isoform is preferentially expressed in small TRPV1-positive nociceptive neurons and only limited expression in IB4-negative and trkA-positive neurons. Cav2.3e mRNA expression is associated with a functionally identified SNX-482-sensitive R-type calcium channel component in these neurons.

Cav2.3e Is the Major Cav2.3 Isoform in TG Neurons—R-type currents, which may result from Cav2.3, are found in most neurons, such as neocortical and striatal neurons (36), CA1 neurons (37), dentate granule cells, cerebellar granule neurons (18), and sensory neurons (24). The Cav2.3 transcripts are widely expressed throughout the brain, as shown by in situ hybridization techniques (38). The full-length cDNA from human Cav2.3d with a genomic fragment from the human genome (GenBankTM accession number NT_004552.7) revealed the intron-exon structure of the Cav2.3 gene, which is composed of 49 exons including a 58-bp 3'-noncoding segment. Exon 19 encodes insert I in the II–III loop, and exon 45 encodes insert III in the carboxylterminal region, and the 21-bp insert II is located within exon 20. Six isoforms (Cav2.3a to Cav2.3f) have been reported in mammalian tissues, with the neuronal Cav2.3c and the endocrine Cav2.3e being the predominant isoforms detected in vivo. Although Cav2.3a has been detected in rat cerebellar granule cells (18), Cav2.3e was initially identified in the rat and human kidney, insulinoma cell line INS-1 cells, and the islets of Langerhans (19). In this study, we found that TG neurons express only two Cav2.3 isoforms, Cav2.3a and Cav2.3e. Of these two isoforms, Cav2.3e, which is known to be a major endocrine Cav2.3 isoform (19), was the predominant isoform in TG neurons. It is not clear at this moment how Cav2.3e regulates neurotransmitter release from nociceptive nerve terminals as well as insulin secretion from islets of Langerhans. However, it is possible that Cav2.3e might be an important molecular mediator, which is involved in both neurotransmitter release and hormone secretion.

Several previous studies support that R-type calcium channels are crucial for nociception. R-type calcium channels are located at primary synapses (39) and contribute to neurotransmitter release and presynaptic plasticity (40). {alpha}lE-/- mice showed reduced response to somatic inflammatory pain (21). Based on our results, Cav2.3e might be the Cav2.3 isoform responsible for the nociception mediated by Cav2.3. Different Cav2.3 isoforms are known to display distinct biophysical properties (41, 42). They might have distinct interactions with specific proteins/modulators.

Physiological Implications—Sensory neurons expressing Cav2.3e exhibited several principal characteristics of nociceptors. First, Cav2.3e was preferentially present in small diameter neurons in the rat TG. Second, Cav2.3e expression was restricted to trkA-positive/IB4-negative nociceptors. Third, Cav2.3e was the predominant isoform in TRPV1-positive neurons. Thus, Cav2.3e is likely to play an important role in nociceptive neurotransmission. Likewise, a Cav2.2 isoform, preferentially present in neurons containing nociceptive markers, TRPV1 and NaV1.8, has been demonstrated to contribute to pain transduction in nociceptive neurons (43).

It is interesting to note that Cav2.3e expression was predominant in trkA-positive/IB4-negative and TRPV1-positive nociceptors. It has been suggested that IB4-positive and IB4-negative populations have different physiological roles in acute and chronic pain conditions (44). Given that IB4-negative neurons contain neuropeptides (30), Cav2.3e might be involved in secretion of calcitonin gene-related neuropeptide and substance P. TRPV1 is a well known nociceptor marker that is preferentially expressed in subpopulations of small nociceptive sensory neurons and plays an important role in thermal nociception and inflammatory hyperalgesia (45). The preferential expression of Cav2.3e in TRPV1-positive nociceptors provides further support for a potential role for Cav2.3e in the transmission of pain. The exact functional role of unique Cav2.3e expression in nociceptive neurons remains to be elucidated.

Correlation between a Functionally Identified R-type Channel Component and Cav2.3e Expression—We verified the molecular nature of R-type calcium channel components by a combination of single cell RT-PCR and functional analyses, including calcium imaging and whole-cell patch clamp recording. A subpopulation of sensory neurons tested definitely showed R-type calcium channel components as demonstrated by residual calcium transients or currents in the presence of L-, N-, and P/Q-type blockers. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recording revealed that Cav2.3e was present in the IB4-negative nociceptive neurons with SNX-482-sensitive R-type calcium channel components, suggesting a correlation between R-type channel and Cav2.3e expression in these neurons. However, there should be another calcium channel in the other subset of IB4-positive neurons, which generates R-type calcium channel components. Previous studies have demonstrated that there may be non-Cav2.3 R-type current in dorsal root ganglion neurons (15, 24). In line with these findings, using the combined approach of antisense strategy and electrophysiology with pharmacological tool, the existence of a non-Cav2.3 R-type current that is resistant to SNX-482 has been reported (41, 42). The molecular identity of this channel remains to be determined.

In summary, this study provides the first evidence of expression of a nociceptor-specific Cav2.3 calcium channel isoform in sensory neurons. Cav2.3e in nociceptive neurons may be a potential target for the treatment of pain.


   FOOTNOTES
 
* This work was supported by MOEHRD Grant KRF-2003-003-E00207 from the Korea Research Foundation, Grant R01-2004-000-10384-0 from the Basic Research Program of the Korea Science and Engineering Foundation, and Grant M103KV010015-06K2201-01510 from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. 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

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 82-2-740-8656; Fax: 82-2-762-5107; E-mail: odolbae{at}snu.ac.kr.

3 The abbreviations used are: TG, trigeminal ganglion; BSS, balanced salt solution; IB4, isolectin B4; RT, reverse transcription; trkA, tyrosine kinase A; TRPV1, transient receptor potential vanilloid 1; VACCs, voltage-activated calcium channels; {omega}-Aga, {omega}-Agatoxin-IVA; {omega}-CgTx, {omega}-conotoxin-GVIA; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sessle, B. J. (2002) J. Dent. Educ. 66, 108-112[Abstract]
  2. Ji, R. R., and Woolf, C. J. (2001) Neurobiol. Dis. 8, 1-10[CrossRef][Medline] [Order article via Infotrieve]
  3. Vanegas, H., and Schaible, H. (2000) Pain 85, 9-18[CrossRef][Medline] [Order article via Infotrieve]
  4. Yaksh, T. L. (2006) J. Pain. 7, Suppl. 1 13-30
  5. Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521-555[CrossRef][Medline] [Order article via Infotrieve]
  6. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233[CrossRef][Medline] [Order article via Infotrieve]
  7. Williams, M. E., Feldman, D. H., McCue, A. F., Brenner, R., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Neuron 8, 71-84[CrossRef][Medline] [Order article via Infotrieve]
  8. Bourinet, E., Soong, T. W., Sutton, K., Slaymaker, S., Mathews, E., Monteil, A., Zamponi, G. W., Nargeot, J., and Snutch, T. P. (1999) Nat. Neurosci. 2, 407-415[CrossRef][Medline] [Order article via Infotrieve]
  9. Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Science 257, 389-395[Abstract/Free Full Text]
  10. Zhang, J. F., Randall, A. D., Ellinor, P. T., Horne, W. A., Sather, W. A., Tanabe, T., Schwarz, T. L., and Tsien, R. W. (1993) Neuropharmacology 32, 1075-1088[CrossRef][Medline] [Order article via Infotrieve]
  11. Lambert, R. C., McKenna, F., Maulet, Y., Talley, E. M., Bayliss, D. A., Cribbs, L. L., Lee, J. H., Perez-Reyes, E., and Feltz, A. (1998) J. Neurosci. 18, 8605-8613[Abstract/Free Full Text]
  12. Perez-Reyes, E., Cribbs, L. L., Daud, A., Lacerda, A. E., Barclay, J., Williamson, M. P., Fox, M., Rees, M., and Lee, J. H. (1998) Nature 391, 896-900[CrossRef][Medline] [Order article via Infotrieve]
  13. Dunlap, K., Luebke, J. I., and Turner, T. J. (1994) Science 266, 828-831[Free Full Text]
  14. Williams, M. E., Marubio, L. M., Deal, C. R., Hans, M., Brust, P. F., Philipson, L. H., Miller, R. J., Johnson, E. C., Harpold, M. M., and Ellis, S. B. (1994) J. Biol. Chem. 269, 22347-22357[Abstract/Free Full Text]
  15. Wilson, S. M., Toth, P. T., Oh, S. B., Gillard, S. E., Volsen, S., Ren, D., Philipson, L. H., Lee, E. C., Fletcher, C. F., Tessarollo, L., Copeland, N. G., Jenkins, N. A., and Miller, R. J. (2000) J. Neurosci. 20, 8566-8571[Abstract/Free Full Text]
  16. Lee, S. C., Choi, S., Lee, T., Kim, H. L., Chin, H., and Shin, H. S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3276-3281[Abstract/Free Full Text]
  17. Schneider, T., Wei, X., Olcese, R., Costantin, J. L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., and Crawford, G. D. (1994) Receptors Channels 2, 255-270[Medline] [Order article via Infotrieve]
  18. Schramm, M., Vajna, R., Pereverzev, A., Tottene, A., Klockner, U., Pietrobon, D., Hescheler, J., and Schneider, T. (1999) Neuroscience 92, 565-575[CrossRef][Medline] [Order article via Infotrieve]
  19. Vajna, R., Schramm, M., Pereverzev, A., Arnhold, S., Grabsch, H., Klockner, U., Perez-Reyes, E., Hescheler, J., and Schneider, T. (1998) Eur. J. Biochem. 257, 274-285[Medline] [Order article via Infotrieve]
  20. Pereverzev, A., Leroy, J., Krieger, A., Malecot, C. O., Hescheler, J., Pfitzer, G., Klockner, U., and Schneider, T. (2002) Mol. Cell. Neurosci. 21, 352-365[CrossRef][Medline] [Order article via Infotrieve]
  21. Saegusa, H., Kurihara, T., Zong, S., Minowa, O., Kazuno, A., Han, W., Matsuda, Y., Yamanaka, H., Osanai, M., Noda, T., and Tanabe, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6132-6137[Abstract/Free Full Text]
  22. Murakami, M., Suzuki, T., Nakagawasai, O., Murakami, H., Murakami, S., Esashi, A., Taniguchi, R., Yanagisawa, T., Tan-No, K., Miyoshi, I., Sasano, H., and Tadano, T. (2001) Brain Res. 903, 231-236[CrossRef][Medline] [Order article via Infotrieve]
  23. Yusaf, S. P., Goodman, J., Pinnock, R. D., Dixon, A. K., and Lee, K. (2001) Neurosci. Lett. 311, 137-141[CrossRef][Medline] [Order article via Infotrieve]
  24. Ikeda, M., and Matsumoto, S. (2003) Life Sci. 73, 1175-1187[CrossRef][Medline] [Order article via Infotrieve]
  25. Yang, B. H., Piao, Z. G., Kim, Y. B., Lee, C. H., Lee, J. K., Park, K., Kim, J. S., and Oh, S. B. (2003) J. Dent. Res. 82, 781-785[Abstract/Free Full Text]
  26. Silbert, S. C., Beacham, D. W., and McCleskey, E. W. (2003) J. Neurosci. 23, 34-42[Abstract/Free Full Text]
  27. Murray, S. S., and Cheema, S. S. (2003) J. Anat. 202, 227-238[CrossRef][Medline] [Order article via Infotrieve]
  28. Stucky, C. L., and Lewin, G. R. (1999) J. Neurosci. 19, 6497-6505[Abstract/Free Full Text]
  29. Murakami, M., Nakagawasai, O., Suzuki, T., Mobarakeh, I. I., Sakurada, Y., Murata, A., Yamadera, F., Miyoshi, I., Yanai, K., Tan-No, K., Sasano, H., Tadano, T., and Iijima, T. (2004) Brain Res. 1024, 122-129[CrossRef][Medline] [Order article via Infotrieve]
  30. Snider, W. D., and McMahon, S. B. (1998) Neuron 20, 629-632[CrossRef][Medline] [Order article via Infotrieve]
  31. Nagy, J. I., and Hunt, S. P. (1982) Neuroscience 7, 89-97[CrossRef][Medline] [Order article via Infotrieve]
  32. Michael, G. J., Averill, S., Nitkunan, A., Rattray, M., Bennett, D. L., Yan, Q., and Priestley, J. V. (1997) J. Neurosci. 17, 8476-8490[Abstract/Free Full Text]
  33. Bradbury, E. J., Burnstock, G., and McMahon, S. B. (1998) Mol. Cell. Neurosci. 12, 256-268[CrossRef][Medline] [Order article via Infotrieve]
  34. Caterina, M. J., and Julius, D. (2001) Annu. Rev. Neurosci. 24, 487-517[CrossRef][Medline] [Order article via Infotrieve]
  35. McCleskey, E. W., and Gold, M. S. (1999) Annu. Rev. Physiol. 61, 835-856[CrossRef][Medline] [Order article via Infotrieve]
  36. Foehring, R. C., Mermelstein, P. G., Song, W. J., Ulrich, S., and Surmeier, D. J. (2000) J. Neurophysiol. 84, 2225-2236[Abstract/Free Full Text]
  37. Ishibashi, H., Rhee, J. S., and Akaike, N. (1995) Neuroreport 6, 1621-1624[Medline] [Order article via Infotrieve]
  38. Niidome, T., Kim, M. S., Friedrich, T., and Mori, Y. (1992) FEBS Lett. 308, 7-13[CrossRef][Medline] [Order article via Infotrieve]
  39. Brown, S. P., Safo, P. K., and Regehr, W. G. (2004) J. Neurosci. 24, 5623-5631[Abstract/Free Full Text]
  40. Dietrich, D., Kirschstein, T., Kukley, M., Pereverzev, A., von der Brelie, C., Schneider, T., and Beck, H. (2003) Neuron 39, 483-496[CrossRef][Medline] [Order article via Infotrieve]
  41. Piedras-Renteria, E. S., and Tsien, R. W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7760-7765[Abstract/Free Full Text]
  42. Tottene, A., Volsen, S., and Pietrobon, D. (2000) J. Neurosci. 20, 171-178[Abstract/Free Full Text]
  43. Bell, T. J., Thaler, C., Castiglioni, A. J., Helton, T. D., and Lipscombe, D. (2004) Neuron 41, 127-138[CrossRef][Medline] [Order article via Infotrieve]
  44. Julius, D., and Basbaum, A. I. (2001) Nature 413, 203-210[CrossRef][Medline] [Order article via Infotrieve]
  45. Szallasi, A., and Blumberg, P. M. (1999) Pharmacol. Rev. 51, 159-212[Abstract/Free Full Text]

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